Method for selection and production of tailor-made highly selective and multi-specific targeting entities containing at least two different binding entities and uses thereof

ABSTRACT

Herein is reported a method for producing a bispecific antibody comprising the step of incubating
         (i) an antibody Fab fragment or a scFv antibody comprising within the 20 C-terminal amino acid residues the amino acid sequence LPX1TG (SEQ ID NO: 01),   (ii) a one-armed antibody comprising a full length antibody heavy chain, a full length antibody light chain, and an Fc-heavy chain,
           whereby the full length antibody heavy chain and the full length antibody light chain are cognate antibody chains that thereof forms an antigen binding site,   whereby the full length antibody heavy chain and the Fc-heavy chain are covalently linked to each other via one or more disulfide bonds forming an antibody hinge region, and   whereby the Fc-heavy chain has an oligoglycine amino acid sequence at its N-terminus,   and   
           (iii) a Sortase A enzyme.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of International Application No.PCT/EP2013/063258 having an international filing date of Jun. 25, 2013,the entire contents of which are incorporated herein by reference, andwhich claims benefit under 35 U.S.C. §119 to European Patent ApplicationNo. 12173875.1 filed Jun. 27, 2012.

Sequence Listing

The instant application contains a Sequence Listing submitted viaEFS-Web and hereby incorporated by reference in its entirety. Said ASCIIcopy, created on Dec. 19, 2014, is named P31069_US_C_SeqList.txt, and is87,663 bytes in size.

Herein is reported a method for selecting and producing multispecificentities by using a transpeptidase, such as Sortase A, wherein thespecificities can be chosen independently of each other and the use ofthis method for the generation of novel tailor-made multispecificantibodies.

BACKGROUND OF THE INVENTION

Monoclonal antibodies have a great therapeutic potential and play animportant role in today's medical portfolio. During the last decade, asignificant trend in the pharmaceutical industry has been thedevelopment of monoclonal antibodies (mAbs) and antibody Fc-fusionpolypeptides (crystallizable fragment-fusion polypeptides) astherapeutic agents across diverse clinical settings including oncology,chronic inflammatory diseases, transplantation, infectious diseases,cardiovascular medicine, or ophthalmologic diseases (Carter, J. P.,Nature Reviews Immunology 6 (2006) 343-357; Chan, A. C. and Carter, J.P., Nature Reviews Immunology 10 (2010) 301-316).

The clinical efficiency of a therapeutic antibody relies mainly on twofunctionalities: i) the target-specific binding mediated by theFv-domain, and ii) the immune-mediated effector function such as ADCC(antibody-dependent cell-mediated cytotoxicity), CDC(complement-dependent cytotoxicity), and ADCP (antibody-dependentcellular phagocytosis) which are mediated by the antibody Fc-region. TheFc-region of an immunoglobulin of the IgG class comprises the hingeregion and two constant domains (CH2 and CH3). The Fc-region alsointeracts with the neonatal FcRn receptor and thereby determines thehalf-life of the antibody in vivo. The hinge region is the region atwhich the arms of an antibody molecule form a Y-like structure enablingflexibility in the molecule at this point. The IgG subclass/subclassesdiffer in the number of disulfide bonds and the length of the hingeregion.

The effector functions associated with the Fc-region of an antibody varywith the class and subclass of the antibody and include e.g. binding ofthe antibody via its Fc-region to a specific Fc receptor (FcR) on a cellwhich triggers various biological responses (see e.g. Jiang, X.-R., etal., Nature Reviews Drug Discovery 10 (2011) 101-110; Presta, L. G.,Current Opinion in Immunology 20 (2008) 460-470).

The hinge region of an antibody or of an Fc-region comprising fusionpolypeptide or conjugate is involved in at least a part of theantibody's functions such as antigen binding and Fc-region-mediatedantibody effector functions. Whereas antigen binding (especiallybivalent avid antibody binding) depends on the flexibility, length andspatial orientation of a particular/native hinge region the Fc-regionmediated effector functions are dependent on the class and subclass ofthe antibody. The functional monovalency observed for some human IgG4antibodies in comparison with the bivalency for the other IgG antibodiesis another example showing the involvement of the Fc-region region inantigen binding properties (Salfeld, J. G., Nature Biotechnology 12(2007) 1369-1372; Presta, L. G., Current Opinion in Immunology 20 (2008)460-470).

Levary, D. A., et al., report protein-protein fusion catalyzed bySortase A (PLOS ONE 6 (2011)). Engineering of an anti-epidermal growthfactor receptor antibody to single chain format and labeling by sortaseA-mediated protein ligation is reported by Madej, M. P., et al.(Biotechnol. Bioeng. 109 (2012) 1461-1470). Ta, H. T., et al., reportenzymatic single-chain antibody tagging as a universal approach totargeted molecular imaging and cell homing in cardiovascular diseases(Cir. Res. 109 (2011) 365-373). Popp, M., et al., report making andbreaking peptide bonds—protein engineering using sortase (Angew. Chem.Int. Ed. Eng. 50 (2011) 5024-5032). In WO 2010/087994 methods forligation and uses thereof are reported.

SUMMARY OF THE INVENTION

Herein is reported a method for providing tailor-made, highly specifictherapeutic molecules for the treatment of a disease, such as cancer, ina patient in need of a treatment, whereby the therapeutic molecule isadapted to the characteristics of the disease of the patient and/or tothe genotype/phenotype of the patient.

Such adaptation is achieved by making a tailor-made molecule taking intoaccount the genotype/phenotype of the disease harboring/affected cellsof the patient.

In a first step the genotype/phenotype of the cells (e.g. the presenceand number/quantity of disease-specific cell surface antigens) that areintended to be targeted with the therapeutic molecule is determined.This can be achieved, e.g. by cell imaging techniques such asimmunohistochemical staining (IHC, immunohistochemistry) of patient'scells derived e.g. from blood and/or biopsied material usingfluorescently labeled monospecific (therapeutic or diagnostic)antibodies. Alternatively the genotype/phenotype of the cells can beanalyzed after staining with labeled therapeutic or diagnosticantibodies using FACS-based methods. In vivo imaging techniquesincluding optical imaging, molecular imaging, fluorescence imaging,bioluminescence Imaging, MRI, PET, SPECT, CT, and intravital microscopymay be used also for determination of the genotype/phenotype ofdisease-related cells of a patient. Depending on the determinedgenotype/phenotype of the disease-related cells of a patient atailor-made combination of targeting/binding entities can be/is chosenand are combined in a therapeutic molecule. Such a therapeutic moleculemay be for example a bispecific antibody.

Such tailor-made therapeutic molecules i) will be highly specific, ii)will have a good efficacy, and iii) will induce less side effectscompared to conventionally chosen therapeutics. This can be achieved byendowing the therapeutic molecule with improved targeting and/orimproved tailor-made delivery properties, e.g. for a therapeutic payloadto its intended site of action.

The improved delivery of the therapeutic molecule to its site of action,such as e.g. a cancer cell, can be achieved by a higher/increasedselectivity and/or specificity of the targeted therapeutic moleculecompared to conventionally chosen therapeutic molecules. The therapeuticmolecule comprises at least two entities that specifically bind todifferent antigens (e.g. two different surface markers) or to differentepitopes on the same antigen (e.g. two different epitopes on the samesurface marker).

The increased selectivity and/or specificity of the tailor-madetherapeutic molecule can be achieved by the simultaneous binding of bothtargeting entities to their respective targets/epitopes, i.e. it isachieved by avidity effects. Especially suited is the combination of twobinding entities having a low to medium affinity for their respectivetargets/epitopes. Additionally, off-target binding is greatly reduced orcan even be eliminated completely.

It has been found that tailor-made bispecific targeting and bindingmolecules can be provided using an enzymatic conjugation reactionbetween a first binding entity, such as a darpin domain based bindingentity, an anticalin domain based binding entity, a T-cell receptorfragment like scTCR domain based binding entity, a camel VH domain basedbinding entity, a tenth fibronectin 3 domain based binding entity, atenascin domain based binding entity, a cadherin domain based bindingentity, an ICAM domain based binding entity, a titin domain basedbinding entity, a GCSF-R domain based binding entity, a cytokinereceptor domain based binding entity, a glycosidase inhibitor domainbased binding entity, a superoxide dismutase domain based bindingentity, or an antibody fragment (Fab or scFv fragment), comprising theamino acid sequence LPX1TG (SEQ ID NO: 01, wherein X1 can be any aminoacid residue) in its C-terminal amino acid sequence region and anone-armed antibody fragment (OA-Fc), which comprises a full lengthantibody heavy chain paired with the cognate full length light chain andan antibody heavy chain Fc-region polypeptide with an oligoglycine G_(m)(m=2, or 3, or 4, or 5) at its N-terminus, using the enzyme Sortase A.

It has been found that with the method as reported herein it is possibleto tailor-make e.g. bispecific antibodies specifically directed to twosurface markers found on the surface of a cell, such as a cancer cell.As the binding specificities are individually provided by the startingcomponents it is possible to tailor-make a multispecific targeting andbinding molecule simply by determining the surface markers present on acell, e.g. on a cancer cell, and conjugating the respective antibodyfragments that specifically bind to these surface markers or theirrespective ligands by an enzymatic procedure. As the enzymaticconjugation is performed by the enzyme Sortase A the resultingbispecific antibody is characterized by the presence of the amino acidsequence LPX1TG ((SEQ ID NO: 01, wherein X1 can be any amino acidresidue).

One aspect as reported herein is a method for producing a multispecificbinding molecule comprising the step of incubating

-   -   (i) a first binding entity comprising within the 20 C-terminal        amino acid residues the amino acid sequence LPX1TG (SEQ ID NO:        01, wherein X1 can be any amino acid residue),    -   (ii) an antibody fragment comprising a full length antibody        heavy chain, a full length antibody light chain and an antibody        heavy chain Fc-region polypeptide,    -   whereby the full length antibody heavy chain and the full length        antibody light chain are cognate antibody chains and the pair of        variable domains (VH and VL) thereof forms an antigen binding        site,    -   whereby the full length antibody heavy chain and the antibody        heavy chain Fc-region polypeptide are covalently linked to each        other via one or more disulfide bonds forming an antibody hinge        region, and    -   whereby the antibody heavy chain Fc-region polypeptide has an        oligoglycine G_(m) (m=2, or 3, or 4, or 5) amino acid sequence        at its N-terminus,    -   and    -   (iii) a Sortase A enzyme    -   and thereby producing the multispecific binding molecule.

One aspect as reported herein is a method for producing a multispecificbinding molecule comprising the following steps

-   -   (i) determining the cell surface makers present in a cell        containing sample and i) selecting thereof at least a first cell        surface marker and a second cell surface marker, or ii)        selecting thereof a multitude of cell surface markers        corresponding to the number of binding specificities of the        multispecific binding molecule,    -   (ii) incubating (a) a first binding entity, which specifically        binds to the first cell surface marker or its ligand, and which        comprises within the 20 C-terminal amino acid residues the amino        acid sequence LPX1TG (SEQ ID NO: 01, wherein X1 can be any amino        acid residue), (b) an antibody fragment comprising a full length        antibody heavy chain, a full length antibody light chain and an        antibody heavy chain Fc-region polypeptide, whereby the full        length antibody heavy chain and the full length antibody light        chain are cognate antibody chains and the pair of variable        domains (VH and VL) thereof forms an antigen binding site, which        specifically binds to the second cell surface marker or its        ligand, whereby the full length antibody heavy chain and the        antibody heavy chain Fc-region polypeptide are covalently linked        to each other via one or more disulfide bonds forming an        antibody hinge region, and whereby the antibody heavy chain        Fc-region polypeptide has an oligoglycine G_(m) (m=2, or 3, or        4, or 5) amino acid sequence at its N-terminus, and (c) a        Sortase A enzyme    -   and thereby producing the multispecific binding molecule.

One aspect as reported herein is a method for the selection of at leasttwo binding entities from a collection/library of binding entities whichare assembled in a single multispecific binding molecule by incubating(a) a first binding entity, which specifically binds to a first epitopeor antigen, and which comprises within the 20 C-terminal amino acidresidues the amino acid sequence LPX1TG (SEQ ID NO: 01, wherein X1 canbe any amino acid residue), (b) an antibody fragment comprising a fulllength antibody heavy chain, a full length antibody light chain and anantibody heavy chain Fc-region polypeptide, whereby the full lengthantibody heavy chain and the full length antibody light chain arecognate antibody chains and the pair of variable domains (VH and VL)thereof forms an antigen binding site, which specifically binds to asecond epitope or antigen, whereby the full length antibody heavy chainand the antibody heavy chain Fc-region polypeptide are covalently linkedto each other via one or more disulfide bonds forming an antibody hingeregion, and whereby the antibody heavy chain Fc-region polypeptide hasan oligoglycine G_(m) (m=2, or 3, or 4, or 5) amino acid sequence at itsN-terminus, and (c) a Sortase A enzyme for use as a therapeutic agent.Such an agent has improved targeting/delivery properties.

One aspect as reported herein is a method for producing a bispecificantibody comprising the step of incubating

-   -   (i) an antibody Fab fragment or a scFv antibody comprising        within the 20 C-terminal amino acid residues the amino acid        sequence LPX1TG (SEQ ID NO: 01, wherein X1 can be any amino acid        residue),    -   (ii) an one-armed antibody fragment comprising a full length        antibody heavy chain, a full length antibody light chain, and an        antibody heavy chain Fc-region polypeptide,    -   whereby the full length antibody heavy chain and the full length        antibody light chain are cognate antibody chains complementary        to each other and the pair of variable domains (VH and VL)        thereof forms an antigen binding site,    -   whereby the full length antibody heavy chain and the antibody        heavy chain Fc-region polypeptide are covalently linked to each        other via one or more disulfide bonds forming an antibody hinge        region, and    -   whereby the antibody heavy chain Fc-region polypeptide has an        oligoglycine G_(m) (m=2, or 3, or 4, or 5) amino acid sequence        at its N-terminus,    -   and    -   (iii) a Sortase A enzyme    -   and thereby producing the bispecific antibody.

One aspect as reported herein is a method for producing a bispecificantibody comprising the following steps

-   -   (i) determining the cell surface makers present in a cell        containing sample and selecting thereof at least a first cell        surface marker and a second cell surface marker,    -   (ii) incubating (a) an antibody Fab fragment or a scFv antibody        comprising within the 20 C-terminal amino acid residues the        amino acid sequence LPX1TG (SEQ ID NO: 01, wherein X1 can be any        amino acid residue), whereby the Fab fragment or scFv antibody        specifically binds to the first cell surface marker or its        ligand, (b) an one-armed antibody fragment comprising a full        length antibody heavy chain, a full length antibody light chain,        and an antibody heavy chain Fc-region polypeptide, whereby the        full length antibody heavy chain and the full length antibody        light chain are cognate antibody chains complementary to each        other and the pair of variable domains (VH and VL) thereof forms        an antigen binding site that specifically binds to the second        cell surface marker or its ligand, whereby the full length        antibody heavy chain and the antibody heavy chain Fc-region        polypeptide are covalently linked to each other via one or more        disulfide bonds forming an antibody hinge region, and whereby        the antibody heavy chain Fc-region polypeptide has an        oligoglycine G_(m) (m=2, or 3, or 4, or 5) amino acid sequence        at its N-terminus, and (c) a Sortase A enzyme    -   and thereby producing the bispecific antibody.

One aspect as reported herein is a method for determining a combinationof binding entities for a multispecific binding molecule comprising thefollowing steps

-   -   (i) determining the binding specificity and/or selectivity        and/or affinity and/or effector function and/or in vivo        half-life of a multitude of multispecific binding molecules        whereby in the multitude of multispecific binding molecules each        (possible) combination of binding entities is comprised,    -   and    -   (ii) choosing the multispecific binding molecule with suitable        binding specificity and/or selectivity and/or affinity and/or        effector function and/or in vivo half-life and thereby        determining a combination of antigen binding sites.

One aspect as reported herein is a method for determining a combinationof antigen binding sites comprising the following steps

-   -   (i) determining the binding specificity and/or selectivity        and/or affinity and/or effector function and/or in vivo        half-life of a multitude of bispecific antibodies prepared by        combining (a) each member of a first multitude of antibody Fab        fragments or scFv antibody fragments whereby each member        comprises within the 20 C-terminal amino acid residues the amino        acid sequence LPX1TG (SEQ ID NO: 01, wherein X1 can be any amino        acid residue), whereby the Fab fragment or scFv antibody        specifically binds to a first epitope or antigen, with (b) each        member of a multitude of one-armed antibody fragment comprising        a full length antibody heavy chain, a full length antibody light        chain, and an antibody heavy chain Fc-region polypeptide,        whereby the full length antibody heavy chain and the full length        antibody light chain are cognate antibody chains complementary        to each other and the pair of variable domains (VH and VL)        thereof forms an antigen binding site that specifically binds to        a second epitope or antigen, whereby the full length antibody        heavy chain and the antibody heavy chain Fc-region polypeptide        are covalently linked to each other via one or more disulfide        bonds forming an antibody hinge region, and whereby the antibody        heavy chain Fc-region polypeptide has an oligoglycine G_(m)        (m=2, or 3, or 4, or 5) amino acid sequence at its N-terminus,        and (c) a Sortase A enzyme    -   and    -   (ii) choosing the bispecific antibody with suitable binding        specificity and/or selectivity and/or affinity and/or effector        function and/or in vivo half-life and thereby determining a        combination of antigen binding sites.

In one embodiment the binding entities are independently of each otherselected from a darpin domain based binding entity, an anticalin domainbased binding entity, a T-cell receptor fragment like scTCR domain basedbinding entity, a camel VH domain based binding entity, a tenthfibronectin 3 domain based binding entity, a tenascin domain basedbinding entity, a cadherin domain based binding entity, an ICAM domainbased binding entity, a titin domain based binding entity, a GCSF-Rdomain based binding entity, a cytokine receptor domain based bindingentity, a glycosidase inhibitor domain based binding entity, asuperoxide dismutase domain based binding entity, or antibody fragmentslike Fab or scFv fragments.

In one embodiment of all aspects the multispecific binding molecule is abispecific antibody, and/or the first binding entity is an antibody Fabfragment or a scFv antibody.

In one embodiment the combining is characterized by incubating theantibody Fab fragment or a scFv antibody fragment and the antibodyfragment comprising a full length antibody heavy chain, a full lengthantibody light chain, and an antibody heavy chain Fc-region polypeptide,with a Sortase A enzyme.

In one embodiment the Fab fragment or scFv antibody fragment compriseswithin the 20 C-terminal amino acid residues the amino acid sequenceLPX1TG (SEQ ID NO: 01, wherein X1 can be any amino acid residue).

In one embodiment the full length antibody heavy chain and the fulllength antibody light chain of the one-armed antibody fragment arecognate antibody chains and the pair of variable domains (VH and VL)thereof forms an antigen binding site that specifically binds to thesecond surface marker, whereby the full length antibody heavy chain andthe antibody heavy chain Fc-region polypeptide are covalently linked toeach other via one or more disulfide bonds forming an antibody hingeregion, and the antibody heavy chain Fc-region polypeptide has anoligoglycine G_(m) (m=2, or 3, or 4, or 5) amino acid sequence at itsN-terminus.

In one embodiment of all aspects the antibody Fab fragment or the scFvantibody comprises within the 20 C-terminal amino acid residues theamino acid sequence G.SLPX1TG (SEQ ID NO: 02, wherein X1 can be anyamino acid residue, with n=1, 2 or 3).

In one embodiment of all aspects the antibody Fab fragment or the scFvantibody comprises within the 20 C-terminal amino acid residues theamino acid sequence GSLPX1TGGSGS (SEQ ID NO: 03, wherein X1 can be anyamino acid residue).

In one embodiment of all aspects the antibody Fab fragment or the scFvantibody comprises within the 20 C-terminal amino acid residues theamino acid sequence GGGSLPX1TGGSGS (SEQ ID NO: 04, wherein X1 can be anyamino acid residue).

In one embodiment of all aspects the antibody Fab fragment or the scFvantibody comprises the amino acid sequence X2GSLPX1TGGSGS (SEQ ID NO:05, wherein X1 can be any amino acid residue) within the 20 C-terminalamino acid residues whereby X2 can be any amino acid residue except G.

In one embodiment of all aspects the antibody Fab fragment or the scFvantibody comprises the amino acid sequence G_(n)SLPX1TGGSGSX3 (SEQ IDNO: 06, wherein X1 can be any amino acid residue, with n=1, 2 or 3)within the 20 C-terminal amino acid residues, whereby X3 is an aminoacid sequence tag.

In one embodiment of all aspects the antibody Fab fragment or the scFvantibody comprises the amino acid sequence X2GSLPX1TGGSGSX3 (SEQ ID NO:07, wherein X1 can be any amino acid residue) within the 20 C-terminalamino acid residues, whereby X2 can be any amino acid residue except G,and whereby X3 is an amino acid sequence tag.

In one embodiment of all aspects the antibody heavy chain Fc-regionpolypeptide comprises two glycine residues at its N-terminus.

In one embodiment of all aspects the one-armed antibody fragmentcomprises the amino acid sequence GGCPX4C (SEQ ID NO: 08) at theN-terminus of its heavy chain, whereby X4 is either S or P.

In one embodiment of all aspects X1 is E.

One aspect as reported herein is a multispecific binding moleculeobtained by a method as reported herein.

One aspect is a multispecific binding molecule comprising the amino acidsequence LPX1TG (SEQ ID NO: 01, wherein X1 can be any amino acidresidue) in one of its heavy chains.

In one embodiment the multispecific binding molecule comprises the aminoacid sequence GnSLPX1TG (SEQ ID NO: 02, wherein X1 can be any amino acidresidue, with n=1, 2 or 3) in one of its heavy chains.

In one embodiment the multispecific binding molecule comprises the aminoacid sequence G.SLPX1TGGCPX4C (SEQ ID NO: 09, wherein X1 can be anyamino acid residue, wherein X4 can be S or P, with n=1, 2 or 3) in oneof its heavy chains.

In one embodiment the multispecific binding molecule comprises the aminoacid sequence X2GSLPX1TGGCPX4C (SEQ ID NO: 10, wherein X1 can be anyamino acid residue, wherein X4 can be S or P) in one of its heavychains, whereby X2 can be any amino acid residue except G.

One aspect as reported herein is a bispecific antibody obtained by amethod as reported herein.

One aspect is a bispecific antibody comprising the amino acid sequenceLPX1TG (SEQ ID NO: 01, wherein X1 can be any amino acid residue) in oneof its heavy chains.

In one embodiment the bispecific antibody comprises the amino acidsequence GnSLPX1TG (SEQ ID NO: 02, wherein X1 can be any amino acidresidue, with n=1, 2 or 3) in one of its heavy chains.

In one embodiment the bispecific antibody comprises the amino acidsequence G_(n)SLPX1TGGCPX4C (SEQ ID NO: 09, wherein X1 can be any aminoacid residue, wherein X4 can be S or P, with n=1, 2 or 3) in one of itsheavy chains.

In one embodiment the bispecific antibody comprises the amino acidsequence X2GSLPX1TGGCPX4C (SEQ ID NO: 10, wherein X1 can be any aminoacid residue, wherein X4 can be S or P) in one of its heavy chains,whereby X2 can be any amino acid residue except G.

One aspect as reported herein is a pharmaceutical formulation comprisinga multispecific binding molecule as reported herein.

One aspect as reported herein is the use of a multispecific bindingmolecule as reported herein in the manufacture of a medicament.

In one embodiment the medicament is for the treatment of cancer.

One aspect as reported herein is a method of treating an individualhaving cancer comprising administering to the individual an effectiveamount of a multispecific binding molecule as reported herein.

One aspect as reported herein is a method for destroying cancer cells inan individual comprising administering to the individual an effectiveamount of a multispecific binding molecule as reported herein.

One aspect as reported herein is a pharmaceutical formulation comprisinga bispecific antibody as reported herein.

One aspect as reported herein is the use of a bispecific antibody asreported herein in the manufacture of a medicament.

In one embodiment the medicament is for the treatment of cancer.

One aspect as reported herein is a method of treating an individualhaving cancer comprising administering to the individual an effectiveamount of a bispecific antibody as reported herein.

One aspect as reported herein is a method for destroying cancer cells inan individual comprising administering to the individual an effectiveamount of a bispecific antibody as reported herein. In one embodiment ofall aspects as reported herein the Fc-region is a human Fc-region or avariant thereof.

In one embodiment the human antibody Fc-region is of human IgG1subclass, or of human IgG2 subclass, or of human IgG3 subclass, or ofhuman IgG4 subclass.

In one embodiment the antibody Fc-region is a human antibody Fc-regionof the human IgG1 subclass, or of the human IgG4 subclass.

In one embodiment the human antibody Fc-region comprises a mutation ofthe naturally occurring amino acid residue at least at one of thefollowing amino acid positions 228, 233, 234, 235, 236, 237, 297, 318,320, 322, 329, and/or 331 to a different residue, wherein the residuesin the antibody Fc-region are numbered according to the EU index ofKabat.

In one embodiment the human antibody Fc-region comprises a mutation ofthe naturally occurring amino acid residue at position 329 and at leastone further mutation of at least one amino acid residue selected fromthe group comprising amino acid residues at position 228, 233, 234, 235,236, 237, 297, 318, 320, 322 and 331 to a different residue, wherein theresidues in the Fc-region are numbered according to the EU index ofKabat. The change of these specific amino acid residues results in analtering of the effector function of the Fc-region compared to thenon-modified (wild-type) Fc-region.

In one embodiment the human antibody Fc-region has a reduced affinity tothe human FcγRIIIA, and/or FcγRIIA, and/or FcγRI compared to a conjugatecomprising the corresponding wild-type IgG Fc-region.

In one embodiment the amino acid residue at position 329 in the humanantibody Fc-region is substituted with glycine, or arginine, or an aminoacid residue large enough to destroy the proline sandwich within theFc-region.

In one embodiment the mutation in the human antibody Fc-region of thenaturally occurring amino acid residue is at least one of S228P, E233P,L234A, L235A, L235E, N297A, N297D, P329G, and/or P331S.

In one embodiment the mutation is L234A and L235A if the antibodyFc-region is of human IgG1 subclass, or S228P and L235E if the antibodyFc-region is of human IgG4 subclass.

In one embodiment the antibody Fc-region comprises the mutation P329G.

In one embodiment the antibody Fc-region comprises the mutation T366W inthe first heavy chain Fc-region polypeptide and the mutations T366S,L368A and Y407V in the second heavy chain Fc-region polypeptide, whereinthe numbering is according to the EU index of Kabat.

In one embodiment the antibody Fc-region comprises the mutation S354C inthe first heavy chain Fc-region polypeptide and the mutation Y349C inthe second heavy chain Fc-region polypeptide.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION

I. Definitions

In the present specification and claims the numbering of the residues inan immunoglobulin heavy chain Fc-region is that of the EU index of Kabat(Kabat, E. A., et al., Sequences of Proteins of Immunological Interest,5th ed., Public Health Service, National Institutes of Health, Bethesda,Md. (1991), NIH Publication 91-3242, expressly incorporated herein byreference).

The term “alteration” denotes the mutation, addition, or deletion of oneor more amino acid residues in a parent amino acid sequence, e.g. of anantibody or fusion polypeptide comprising at least an FcRn bindingportion of an Fc-region, to obtain a variant antibody or fusion polypeptide.

The term “amino acid mutation” denotes a modification in the amino acidsequence of a parent amino acid sequence. Exemplary modificationsinclude amino acid substitutions, insertions, and/or deletions. In oneembodiment the amino acid mutation is a substitution. The term “aminoacid mutations at the position” denotes the substitution or deletion ofthe specified residue, or the insertion of at least one amino acidresidue adjacent the specified residue. The term “insertion adjacent toa specified residue” denotes the insertion within one to two residuesthereof. The insertion may be N-terminal or C-terminal to the specifiedresidue.

The term “amino acid substitution” denotes the replacement of at leastone amino acid residue in a predetermined parent amino acid sequencewith a different “replacement” amino acid residue. The replacementresidue or residues may be a “naturally occurring amino acid residue”(i.e. encoded by the genetic code) and selected from the groupconsisting of: alanine (Ala); arginine (Arg); asparagine (Asn); asparticacid (Asp); cysteine (Cys); glutamine (Gln); glutamic acid (Glu);glycine (Gly); histidine (His); isoleucine (Ile): leucine (Leu); lysine(Lys); methionine (Met); phenylalanine (Phe); proline (Pro); serine(Ser); threonine (Thr); tryptophan (Trp); tyrosine (Tyr); and valine(Val). In one embodiment the replacement residue is not cysteine.Substitution with one or more non-naturally occurring amino acidresidues is also encompassed by the definition of an amino acidsubstitution herein. A “non-naturally occurring amino acid residue”denotes a residue, other than those naturally occurring amino acidresidues listed above, which is able to covalently bind adjacent aminoacid residues(s) in a polypeptide chain. Examples of non-naturallyoccurring amino acid residues include norleucine, ornithine, norvaline,homoserine, aib and other amino acid residue analogues such as thosedescribed in Ellman, et al., Meth. Enzym. 202 (1991) 301-336. Togenerate such non-naturally occurring amino acid residues, theprocedures of Noren, et al. (Science 244 (1989) 182) and/or Ellman, etal. (supra) can be used. Briefly, these procedures involve chemicallyactivating a suppressor tRNA with a non-naturally occurring amino acidresidue followed by in vitro transcription and translation of the RNA.Non-naturally occurring amino acids can also be incorporated intopeptides via chemical peptide synthesis and subsequent fusion of thesepeptides with recombinantly produced polypeptides, such as antibodies orantibody fragments.

The term “amino acid insertion” denotes the incorporation of at leastone additional amino acid residue into a predetermined parent amino acidsequence. While the insertion will usually consist of the insertion ofone or two amino acid residues, the present application contemplateslarger “peptide insertions”, e.g. insertion of about three to about fiveor even up to about ten amino acid residues. The inserted residue(s) maybe naturally occurring or non-naturally occurring as defined above.

The term “amino acid deletion” denotes the removal of at least one aminoacid residue at a predetermined position in an amino acid sequence.

Within this application whenever an amino acid alteration is mentionedit is a deliberated amino acid alteration and not a random amino acidmodification.

The term “amino acid sequence tag” denotes a sequence of amino acidresidues connected to each other via peptide bonds that has specificbinding properties. In one embodiment the amino acid sequence tag is anaffinity or purification tag. In one embodiment the amino acid sequencetag is selected from Arg-tag, His-tag, Flag-tag, 3xFlag-tag, Strep-tag,Nano-tag, SBP-tag, c-myc-tag, S-tag, calmodulin-binding-peptide,cellulose-binding-domain, chitin-binding-domain, GST-tag, or MBP-tag. Inone embodiment the amino acid sequence tag is selected from SEQ ID NO:11 (RRRRR), or SEQ ID NO: 12 (RRRRRR), or SEQ ID NO: 13 (HHHHHH), or SEQID NO: 14 (KDHLIHNVHKEFHAHAHNK), or SEQ ID NO: 15 (DYKDDDDK), or SEQ IDNO: 16 (DYKDHDGDYKDHDIDYKDDDDK), or SEQ ID NO: 17 (AWRHPQFGG), or SEQ IDNO: 18 (WSHPQFEK), or SEQ ID NO: 19 (MDVEAWLGAR), or SEQ ID NO: 20(MDVEAWLGARVPLVET), or SEQ ID NO: 21(MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP), or SEQ ID NO: 22 (EQKLISEEDL),or SEQ ID NO: 23 (KETAAAKFERQHMDS), or SEQ ID NO: 24(KRRWKKNFIAVSAANRFKKISSSGAL), or SEQ ID NO: 25 (cellulose bindingdomain), or SEQ ID NO: 26 (cellulose binding domain), or SEQ ID NO: 27(TNPGVSAWQVNTAYTAGQLVTYNGKTYKCLQPHTSLAGWEP SNVPALWQLQ), or SEQ ID NO: 28(GST-tag), or SEQ ID NO: 29 (MBP-tag).

The term “antibody fragment” denotes a molecule other than a full lengthantibody that comprises a portion of a full length antibody that bindsthe antigen to which the intact antibody binds. Examples of antibodyfragments include but are not limited to Fv, Fab, Fab′, Fab′-SH,F(ab′)₂, diabodies, linear antibodies, single-chain antibody molecules(e.g. scFv), and multispecific antibodies formed from antibodyfragments.

Antibody fragments include, but are not limited to, Fab, Fab′, Fab′-SH,F(ab′)₂, Fv, and scFv fragments, and other fragments described below.For a review of certain antibody fragments, see Hudson, P. J., et al.,Nat. Med. 9 (2003) 129-134. For a review of scFv fragments, see, e.g.,Plueckthun, A., In: The Pharmacology of Monoclonal Antibodies, vol. 113,Rosenburg and Moore (eds.), Springer-Verlag, New York (1994), pp.269-315; see also WO 93/16185; U.S. Pat. No. 5,571,894 and U.S. Pat. No.5,587,458. For discussion of Fab and F(ab′)₂ fragments comprisingsalvage receptor binding epitope residues and having increased in vivohalf-life, see U.S. Pat. No. 5,869,046.

Diabodies are antibody fragments with two antigen-binding sites that maybe bivalent or bispecific. See, for example, EP 0 404 097; WO1993/01161; Hudson, P. J., et al., Nat. Med. 9 (2003) 129-134; andHolliger, P., et al., Proc. Natl. Acad. Sci. USA 90 (1993) 6444-6448.Triabodies and tetrabodies are also described in Hudson, P. J., et al.,Nat. Med. 9 (2003) 129-134).

Single-domain antibodies are antibody fragments comprising all or aportion of the heavy chain variable domain or all or a portion of thelight chain variable domain of an antibody. In certain embodiments, asingle-domain antibody is a human single-domain antibody (Domantis,Inc., Waltham, Mass.; see, e.g., U.S. Pat. No. 6,248,516 B1).

Antibody fragments can be made by various techniques, including but notlimited to proteolytic digestion of an intact antibody as well asproduction by recombinant host cells (e.g. E. coli or phage), asdescribed herein.

The term “bispecific antibody” denotes an antigen binding molecule thatcan specifically bind to a first antigen or epitope and to a secondantigen or epitope, whereby the first antigen or epitope are differentfrom the second antigen or epitope.

Bispecific antibody formats are described e.g. in WO 2009/080251, WO2009/080252, WO 2009/080253, WO 2009/080254, WO 2010/112193, WO2010/115589, WO 2010/136172, WO 2010/145792, and WO 2010/145793.

The term “antibody-dependent cell-mediated cytotoxicity”, short “ADCC”,denotes a cell-mediated reaction in which non-antigen specific cytotoxiccells that express FcRs (e.g. natural killer cells (NK cells),neutrophils, and macrophages) recognize a target cell by binding toimmunoglobulin Fc-region and subsequently cause lysis of the targetcell. The primary cells for mediating ADCC, NK cells, express FcγRIIIonly, whereas monocytes express FcγRI, FcγRII and FcγRIII. FcRexpression on hematopoietic cells is summarized in Table 3 on page 464of Ravetch and Kinet, Annu Rev. Immunol. 9 (1991) 457-492.

The term “antibody-dependent cellular phagocytosis”, short “ADCP”,denotes a process by which antibody-coated cells are internalized,either in whole or in part, by phagocytic immune cells (e.g.macrophages, neutrophils, or dendritic cells) that bind to animmunoglobulin Fc-region.

The term “binding to an Fc receptor” denotes the binding of an Fc-regionto an Fc receptor in, for example, a BIAcore® assay (Pharmacia BiosensorAB, Uppsala, Sweden).

In the BIAcore® assay the Fc receptor is bound to a surface and bindingof the analyte, e.g. an Fc-region comprising fusion polypeptide or anantibody, is measured by surface plasmon resonance (SPR). The affinityof the binding is defined by the terms ka (association constant: rateconstant for the association of the Fc-region fusion polypeptide orconjugate to form an Fc-region/Fc receptor complex), kd (dissociationconstant; rate constant for the dissociation of the Fc-region fusionpolypeptide or conjugate from an Fc-region/Fc receptor complex), and KD(kd/ka). Alternatively, the binding signal of a SPR sensorgram can becompared directly to the response signal of a reference, with respect tothe resonance signal height and the dissociation behaviors.

The term “C1q” denotes a polypeptide that includes a binding site forthe Fc-region of an immunoglobulin. C1q together with two serineproteases, C1r and C1s, forms the complex C1, the first component of thecomplement dependent cytotoxicity (CDC) pathway. Human C1q can bepurchased commercially from, e.g. Quidel, San Diego, Calif.

The term “CH2 domain” denotes the part of an antibody heavy chainpolypeptide that extends approximately from EU position 231 to EUposition 340 (EU numbering system according to Kabat). In one embodimenta CH2 domain has the amino acid sequence of

APELLGGP SVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQESTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAK (SEQ ID NO:30). The CH2 domain is unique in that it is not closely paired withanother domain. Rather, two N-linked branched carbohydrate chains areinterposed between the two CH2 domains of an intact native Fc-region. Ithas been speculated that the carbohydrate may provide a substitute forthe domain-domain pairing and help stabilize the CH2 domain. Burton,Mol. Immunol. 22 (1985) 161-206.

The term “CH3 domain” denotes the part of an antibody heavy chainpolypeptide that extends approximately from EU position 341 to EUposition 446. In one embodiment the CH3 domain has the amino acidsequence of GQPREPQVYTLPP SRDELTKNQVS LTCLVKGFYP SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPG (SEQ ID NO:31).

The term “class” of an antibody denotes the type of constant domain orconstant region possessed by its heavy chain. There are five majorclasses of antibodies in humans: IgA, IgD, IgE, IgG, and IgM, andseveral of these may be further divided into subclasses (isotypes),e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constantdomains that correspond to the different classes of immunoglobulins arecalled α, δ, δ, γ, and μ, respectively.

The term “complement-dependent cytotoxicity”, short “CDC”, denotes amechanism for inducing cell death in which an Fc-region of atarget-bound Fc-region fusion polypeptide or conjugate activates aseries of enzymatic reactions culminating in the formation of holes inthe target cell membrane. Typically, antigen-antibody complexes such asthose on antibody-coated target cells bind and activate complementcomponent C1q which in turn activates the complement cascade leading totarget cell death. Activation of complement may also result indeposition of complement components on the target cell surface thatfacilitate ADCC or ADCP by binding complement receptors (e.g., CR3) onleukocytes.

The term “effector function” denotes those biological activitiesattributable to the Fc-region of an antibody, which vary with theantibody subclass. Examples of antibody effector functions include: C1qbinding and complement dependent cytotoxicity (CDC); Fc receptorbinding; antibody-dependent cell-mediated cytotoxicity (ADCC);phagocytosis (ADCP); down regulation of cell surface receptors (e.g.B-cell receptor); and B-cell activation. Such function can be effectedby, for example, binding of an Fc-region to an Fc receptor on an immunecell with phagocytic or lytic activity, or by binding of an Fc-region tocomponents of the complement system.

An “effective amount” of an agent, e.g., a pharmaceutical formulation,refers to an amount effective, at dosages and for periods of timenecessary, to achieve the desired therapeutic or prophylactic result.

The term “reduced effector function” denotes a reduction of a specificeffector function associated with a molecule, like for example ADCC orCDC, in comparison to a control molecule (for example a polypeptide witha wild-type Fc-region) by at least 20%. The term “strongly reducedeffector function” denotes a reduction of a specific effector functionassociated with a molecule, like for example ADCC or CDC, in comparisonto a control molecule by at least 50%.

The term “Fc-region” denotes the C-terminal region of an immunoglobulin.The Fc-region is a dimeric molecule comprising two disulfide-linkedantibody heavy chain fragments (heavy chain Fc-region polypeptidechains). An Fc-region can be generated by papain digestion, or IdeSdigestion, or trypsin digestion of an intact (full length) antibody orcan be produced recombinantly.

The Fc-region obtainable from a full length antibody or immunoglobulincomprises at least residues 226 (Cys) to the C-terminus of the fulllength heavy chain and, thus, comprises a part of the hinge region andtwo or three constant domains, i.e. a CH2 domain, a CH3 domain, and anadditional/extra CH4 domain on IgE and IgM class antibodies. It is knownfrom U.S. Pat. No. 5,648,260 and U.S. Pat. No. 5,624,821 that themodification of defined amino acid residues in the Fc-region results inphenotypic effects.

The formation of the dimeric Fc-region comprising two identical ornon-identical antibody heavy chain fragments is mediated by thenon-covalent dimerization of the comprised CH3 domains (for involvedamino acid residues see e.g. Dall'Acqua, Biochem. 37 (1998) 9266-9273).The Fc-region is covalently stabilized by the formation of disulfidebonds in the hinge region (see e.g. Huber, et al., Nature 264 (1976)415-420; Thies, et al., J. Mol. Biol. 293 (1999) 67-79). Theintroduction of amino acid residue changes within the CH3 domain inorder to disrupt the dimerization of CH3-CH3 domain interactions do notadversely affect the neonatal Fc receptor (FcRn) binding due to thelocation of the CH3-CH3-domain dimerization involved residues arelocated on the inner interface of the CH3 domain, whereas the residuesinvolved in Fc-region-FcRn interaction are located on the outside of theCH2-CH3 domain.

The residues associated with effector functions of an Fc-region arelocated in the hinge region, the CH2, and/or the CH3 domain asdetermined for a full length antibody molecule. The Fc-regionassociated/mediated functions are:

-   -   (i) antibody-dependent cellular cytotoxicity (ADCC),    -   (ii) complement (Clq) binding, activation and        complement-dependent cytotoxicity (CDC),    -   (iii) phagocytosis/clearance of antigen-antibody complexes,    -   (iv) cytokine release in some instances, and    -   (v) half-life/clearance rate of antibody and antigen-antibody        complexes.

The Fc-region associated effector functions are initiated by theinteraction of the Fc-region with effector function specific moleculesor receptors. Mostly antibodies of the IgG1 subclass can effect receptoractivation, whereas antibodies of the IgG2 and IgG4 subclasses do nothave effector function or have limited effector function.

The effector function eliciting receptors are the Fc receptor types (andsub-types) FcγRI, FcγRII and FcγRIII. The effector functions associatedwith an IgG1 subclass can be reduced by introducing specific amino acidchanges in the lower hinge region, such as L234A and/or L235A, which areinvolved in FcγR and C1q binding. Also certain amino acid residues,especially located in the CH2 and/or CH3 domain, are associated with thecirculating half-life of an antibody molecule or an Fc-region fusionpolypeptide in the blood stream. The circulatory half-life is determinedby the binding of the Fc-region to the neonatal Fc receptor (FcRn).

The sialyl residues present on the Fc-region glycostructure are involvedin anti-inflammatory mediated activity of the Fc-region (see e.g.Anthony, R. M., et al., Science 320 (2008) 373-376).

The numbering of the amino acid residues in the constant region of anantibody is made according to the EU index of Kabat (Kabat, E. A., etal., Sequences of Proteins of Immunological Interest, 5th ed., PublicHealth Service, National Institutes of Health, Bethesda, Md. (1991), NIHPublication 91 3242).

The term “human Fc-region” denotes the C-terminal region of animmunoglobulin heavy chain of human origin that contains at least a partof the hinge region, the CH2 domain and the CH3 domain. In oneembodiment, a human IgG antibody heavy chain Fc-region extends fromabout Glu216, or from about Cys226, or from about Pro230, to thecarboxyl-terminus of the heavy chain. However, the C-terminal lysine(Lys447) of the antibody Fc-region may or may not be present.

The term “variant Fc-region” denotes an amino acid sequence whichdiffers from that of a “native” or “wild-type” Fc-region amino acidsequence by virtue of at least one “amino acid alteration/mutation”. Inone embodiment the variant Fc-region has at least one amino acidmutation compared to a native Fc-region or to the Fc-region of a parentpolypeptide, e.g. from about one to about ten amino acid mutations, andin one embodiment from about one to about five amino acid mutations in anative Fc-region or in the Fc-region of the parent polypeptide. In oneembodiment the (variant) Fc-region has at least about 80% homology witha wild-type Fc-region and/or with an Fc-region of a parent polypeptide,and in one embodiment the variant Fc-region has least about 90%homology, in one embodiment the variant Fc-region has at least about 95%homology.

The variant Fc-regions as reported herein are defined by the amino acidalterations that are contained. Thus, for example, the term P329Gdenotes a variant Fc-region with the mutation of proline to glycine atamino acid position 329 relative to the parent (wild-type) Fc-region.The identity of the wild-type amino acid may be unspecified, in whichcase the aforementioned variant is referred to as 329G. For allpositions discussed in the present invention, numbering is according tothe EU index. The EU index or EU index as in Kabat or EU numberingscheme refers to the numbering of the EU antibody (Edelman, et al.,Proc. Natl. Acad. Sci. USA 63 (1969) 78-85, hereby entirely incorporatedby reference.) The alteration can be an addition, deletion, or mutation.The term “mutation” denotes a change to naturally occurring amino acidsas well as a change to non-naturally occurring amino acids, see e.g.U.S. Pat. No. 6,586,207, WO 98/48032, WO 03/073238, US 2004/0214988, WO2005/35727, WO 2005/74524, Chin, J. W., et al., J. Am. Chem. Soc. 124(2002) 9026-9027; Chin, J. W. and Schultz, P. G., ChemBioChem 11 (2002)1135-1137; Chin, J. W., et al., PICAS United States of America 99 (2002)11020-11024; and, Wang, L. and Schultz, P. G., Chem. (2002) 1-10 (allentirely incorporated by reference herein).

A polypeptide chain of a wild-type human Fc-region of the IgG1 subclasshas the following amino acid sequence:

(SEQ ID NO: 32) CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

A polypeptide chain of a variant human Fc-region of the IgG1 subclasswith the mutations L234A, L235A has the following amino acid sequence:

(SEQ ID NO: 33) CPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

A polypeptide chain of a variant human Fc-region of the IgG1 subclasswith a T366S, 368A, and Y407V mutation has the following amino acidsequence:

(SEQ ID NO: 34) CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

A polypeptide chain of a variant human Fc-region of the IgG1 subclasswith a T366W mutation has the following amino acid sequence:

(SEQ ID NO: 35) CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

A polypeptide chain of a variant human Fc-region of the IgG1 subclasswith a L234A, L235A and T366S, 368A, and Y407V mutation has thefollowing amino acid sequence:

(SEQ ID NO: 36) CPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

A polypeptide chain of a variant human Fc-region of the IgG1 subclasswith a L234A, L235A and T366W mutation has the following amino acidsequence:

(SEQ ID NO: 37) CPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

A polypeptide chain of a variant human Fc-region of the IgG1 subclasswith a P329G mutation has the following amino acid sequence:

(SEQ ID NO: 38) CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

A polypeptide chain of a variant human Fc-region of the IgG1 subclasswith a L234A, L235A and P329G mutation has the following amino acidsequence:

(SEQ ID NO: 39) CPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

A polypeptide chain of a variant human Fc-region of the IgG1 subclasswith a P239G and T366S, 368A, and Y407V mutation has the following aminoacid sequence:

(SEQ ID NO: 40) CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

A polypeptide chain of a variant human Fc-region of the IgG1 subclasswith a P329G and T366W mutation has the following amino acid sequence:

(SEQ ID NO: 41) CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

A polypeptide chain of a variant human Fc-region of the IgG1 subclasswith a L234A, L235A, P329G and T366S, 368A, and Y407V mutation has thefollowing amino acid sequence:

(SEQ ID NO: 42) CPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

A polypeptide chain of a variant human Fc-region of the IgG1 subclasswith a L234A, L235A, P329G and T366W mutation has the following aminoacid sequence:

(SEQ ID NO: 43) CPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

A polypeptide chain of a wild-type human Fc-region of the IgG4 subclasshas the following amino acid sequence:

(SEQ ID NO: 44) CPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

A polypeptide chain of a variant human Fc-region of the IgG4 subclasswith a S228P and L235E mutation has the following amino acid sequence:

(SEQ ID NO: 45) CPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

A polypeptide chain of a variant human Fc-region of the IgG4 subclasswith a S228P, L235E and P329G mutation has the following amino acidsequence:

(SEQ ID NO: 46) CPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLGSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

The term “Fc receptor”, short “FcR”, denotes a receptor that binds to anFc-region.

In one embodiment the FcR is a native sequence human FcR. Moreover, inone embodiment the FcR is an FcR which binds an IgG antibody (an Fcgamma receptor) and includes receptors of the FcγRI, FcγRII, and FcγRIIIsubclasses, including allelic variants and alternatively spliced formsthereof. FcγRII receptors include FcγRIIA (an “activating receptor”) andFcγRIIB (an “inhibiting receptor”), which have similar amino acidsequences that differ primarily in the cytoplasmic domains thereof.Activating receptor FcγRIIA contains an immunoreceptor tyrosine-basedactivation motif (ITAM) in its cytoplasmic domain Inhibiting receptorFcγRIIB contains an immunoreceptor tyrosine-based inhibition motif(ITIM) in its cytoplasmic domain (see e.g. Daëron, M., Annu Rev.Immunol. 15 (1997) 203-234). FcRs are reviewed in Ravetch and Kinet,Annu Rev. Immunol. 9 (1991) 457-492, Capel, et al., Immunomethods 4(1994) 25-34, de Haas, et al., J. Lab. Clin. Med. 126 (1995) 330-341.Other FcRs, including those to be identified in the future, areencompassed by the term “FcR” herein. The term also includes theneonatal receptor, FcRn, which is responsible for the transfer ofmaternal IgGs to the fetus (see e.g. Guyer, et al., J. Immunol. 117(1976) 587; Kim, et al., J. Immunol. 24 (1994) 249).

The term “Fc gamma receptor”, short “FcγR”, denotes any member of thefamily of proteins that bind the IgG antibody Fc-region and is encodedby an FcγR gene. In humans this family includes but is not limited toFcγRI (CD64), including isoforms FcγRIA, FcγRIB, and FcγRIC, FcγRII(CD32), including isoforms FcγRIIA (including allotypes H131 and R131),FcγRIIB (including FcγRIIB-1 and FcγRIIB-2), and FcγRIIC, and FcγRIII(CD16), including isoforms FcγRIIIA (including allotypes V158 and F158)and FcγRIIIB (including allotypes FcγRIIB-NA1 and FcγRIIB-NA2) (see e.g.Jefferis, et al., Immunol. Lett. 82 (2002) 57-65, entirely incorporatedby reference), as well as any undiscovered human FcγRs or FcγR isoformsor allotypes. An FcγR may be from any organism, including but notlimited to humans, mice, rats, rabbits, and monkeys. Mouse FcγRs includebut are not limited to FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16), andFcγRIII-2 (CD16-2), as well as any undiscovered mouse FcγRs or FcγRisoforms or allotypes. The Fc-region-FcγR interaction involved aminoacid residues are 234-239 (lower hinge region), 265-269 (B/C loop),297-299 (D/E loop), and 327-332 (F/G) loop (Sondermann, et al., Nature406 (2000) 267-273). Amino acid mutations that result in a decreasedbinding/affinity for the FcγRI, FcγRIIA, FcγRIIB, and/or FcγRIIIAinclude N297A (concomitantly with a decreased immunogenicity andprolonged half-life binding/affinity) (Routledge, et al.,Transplantation 60 (1995) 847; Friend, et al., Transplantation 68 (1999)1632; Shields, et al., J. Biol. Chem. 276 (2001) 6591-6604), residues233-236 (Ward and Ghetie, Ther. Immunol. 2 (1995) 77; Armour, et al.,Eur. J. Immunol. 29 (1999) 2613-2624). Some exemplary amino acidsubstitutions are described in U.S. Pat. No. 7,355,008 and U.S. Pat. No.7,381,408.

The term “neonatal Fc Receptor”, short “FcRn”, denotes a protein thatbinds the IgG antibody Fc-region and is encoded at least in part by anFcRn gene. The FcRn may be from any organism, including but not limitedto humans, mice, rats, rabbits, and monkeys. As is known in the art, thefunctional FcRn protein comprises two polypeptides, often referred to asthe heavy chain and light chain. The light chain is beta-2-microglobulinand the heavy chain is encoded by the FcRn gene. Unless otherwise notedherein, FcRn or an FcRn protein refers to the complex of FcRn heavychain with beta-2-microglobulin. The interacting amino acid residues ofthe Fc-region with the FcRn are near the junction of the CH2 and CH3domains. The Fc-region-FcRn contact residues are all within a single IgGheavy chain. The involved amino acid residues are 248, 250-257, 272,285, 288, 290-291, 308-311, and 314 (all in the CH2 domain) and aminoacid residues 385-387, 428, and 433-436 (all in the CH3 domain). Aminoacid mutations that result in an increased binding/affinity for the FcRninclude T256A, T307A, E380A, and N434A (Shields, et al., J. Biol. Chem.276 (2001) 6591-6604).

The term “full length antibody” denotes an antibody that has a structureand amino acid sequence substantially identical to a native antibodystructure as well as polypeptides that comprise the Fc-region asreported herein.

The term “full length antibody heavy chain” denotes a polypeptidecomprising in N- to C-terminal direction an antibody variable domain, afirst constant domain, an antibody heavy chain hinge region, a secondconstant domain, and a third constant domain.

The term “antibody heavy chain Fc-region” denotes a polypeptidecomprising an antibody heavy chain hinge region, a first constantdomain, and a second constant domain.

The term “full length antibody light chain” denotes a polypeptidecomprising in N- to C-terminal direction an antibody variable domain anda constant domain.

The term “hinge region” denotes the part of an antibody heavy chainpolypeptide that joins in a wild-type antibody heavy chain the CH1domain and the CH2 domain, e. g. from about position 216 to aboutposition 230 according to the EU number system of Kabat, or from aboutposition 226 to about position 230 according to the EU number system ofKabat. The hinge regions of other IgG subclasses can be determined byaligning with the hinge-region cysteine residues of the IgG1 subclasssequence.

The hinge region is normally a dimeric molecule consisting of twopolypeptides with identical amino acid sequence. The hinge regiongenerally comprises about 25 amino acid residues and is flexibleallowing the antigen binding regions to move independently. The hingeregion can be subdivided into three domains: the upper, the middle, andthe lower hinge domain (see e.g. Roux, et al., J. Immunol. 161 (1998)4083).

The term “lower hinge region” of an Fc-region denotes the stretch ofamino acid residues immediately C-terminal to the hinge region, i.e.residues 233 to 239 of the Fc-region according to the EU numbering ofKabat.

The term “wild-type Fc-region” denotes an amino acid sequence identicalto the amino acid sequence of an Fc-region found in nature. Wild-typehuman Fc-regions include a native human IgG1 Fc-region (non-A and Aallotypes), native human IgG2 Fc-region, native human IgG3 Fc-region,and native human IgG4 Fc-region as well as naturally occurring variantsthereof.

The term “individual” or “subject” denotes a mammal. Mammals include,but are not limited to, domesticated animals (e.g. cows, sheep, cats,dogs, and horses), primates (e.g., humans and non-human primates such asmonkeys), rabbits, and rodents (e.g., mice and rats). In certainembodiments, the individual or subject is a human.

“Percent (%) amino acid sequence identity” with respect to a referencepolypeptide sequence is defined as the percentage of amino acid residuesin a candidate sequence that are identical with the amino acid residuesin the reference polypeptide sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentamino acid sequence identity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)software. Those skilled in the art can determine appropriate parametersfor aligning sequences, including any algorithms needed to achievemaximal alignment over the full length of the sequences being compared.For purposes herein, however, % amino acid sequence identity values aregenerated using the sequence comparison computer program ALIGN-2. TheALIGN-2 sequence comparison computer program was authored by Genentech,Inc., and the source code has been filed with user documentation in theU.S. Copyright Office, Washington D.C., 20559, where it is registeredunder U.S. Copyright Registration No. TXU510087. The ALIGN-2 program ispublicly available from Genentech, Inc., South San Francisco, Calif., ormay be compiled from the source code. The ALIGN-2 program should becompiled for use on a UNIX operating system, including digital UNIXV4.0D. All sequence comparison parameters are set by the ALIGN-2 programand do not vary.

In situations where ALIGN-2 is employed for amino acid sequencecomparisons, the % amino acid sequence identity of a given amino acidsequence A to, with, or against a given amino acid sequence B (which canalternatively be phrased as a given amino acid sequence A that has orcomprises a certain % amino acid sequence identity to, with, or againsta given amino acid sequence B) is calculated as follows:

100 times the fraction X/Y

where X is the number of amino acid residues scored as identical matchesby the sequence alignment program ALIGN-2 in that program's alignment ofA and B, and where Y is the total number of amino acid residues in B. Itwill be appreciated that where the length of amino acid sequence A isnot equal to the length of amino acid sequence B, the % amino acidsequence identity of A to B will not equal the % amino acid sequenceidentity of B to A. Unless specifically stated otherwise, all % aminoacid sequence identity values used herein are obtained as described inthe immediately preceding paragraph using the ALIGN-2 computer program.

The term “pharmaceutical formulation” refers to a preparation which isin such form as to permit the biological activity of an activeingredient contained therein to be effective, and which contains noadditional components which are unacceptably toxic to a subject to whichthe formulation would be administered.

A “pharmaceutically acceptable carrier” refers to an ingredient in apharmaceutical formulation, other than an active ingredient, which isnontoxic to a subject. A pharmaceutically acceptable carrier includes,but is not limited to, a buffer, excipient, stabilizer, or preservative.

The term “phenotype of a patient” denotes the composition of cellsurface receptors in a kind of cells from a patient. The composition canbe a qualitative as well as a quantitative composition. The cells forwhich the genotype is determined/given can be a single cell or a samplecomprising multiple cells.

The term “position” denotes the location of an amino acid residue in theamino acid sequence of a polypeptide. Positions may be numberedsequentially, or according to an established format, for example the EUindex of Kabat for antibody numbering.

The term “altered” FcR binding affinity or ADCC activity denotes apolypeptide that has either enhanced or diminished FcR binding activityand/or ADCC activity compared to a parent polypeptide (e.g. apolypeptide comprising a wild-type Fc-region). The variant polypeptidewhich “has increased binding” to an FcR binds at least one FcR withlower dissociation constant (i.e. better/higher affinity) than theparent or wild-type polypeptide. The polypeptide variant which “hasdecreased binding” to an FcR, binds at least one FcR with higherdissociation constant (i.e. worse/lower affinity) than the parent or awild-type polypeptide. Such variants which display decreased binding toan FcR may possess little or no appreciable binding to an FcR, e.g.,0-20% binding to the FcR compared to a wild-type or parent IgGFc-region.

The polypeptide which binds an FcR with “reduced affinity” in comparisonwith a parent or wild-type polypeptide, is a polypeptide which binds anyone or more of the above identified FcRs with (substantially) reducedbinding affinity compared to the parent polypeptide, when the amounts ofpolypeptide variant and parent polypeptide in the binding assay are(essentially) about the same. For example, the polypeptide variant withreduced FcR binding affinity may display from about 1.15 fold to about100 fold, e.g. from about 1.2 fold to about 50 fold reduction in FcRbinding affinity compared to the parent polypeptide, where FcR bindingaffinity is determined.

The polypeptide comprising a variant Fc-region which “mediatesantibody-dependent cell-mediated cytotoxicity (ADCC) in the presence ofhuman effector cells less effectively” than a parent polypeptide is onewhich in vitro or in vivo is (substantially) less effective at mediatingADCC, when the amounts of variant polypeptide and parent polypeptideused in the assay are (essentially) about the same. Generally, suchvariants will be identified using the in vitro ADCC assay as disclosedherein, but other assays or methods for determining ADCC activity, e.g.in an animal model etc., are contemplated. In one embodiment the variantis from about 1.5 fold to about 100 fold, e.g. from about two fold toabout fifty fold, less effective at mediating ADCC than the parent, e.g.in the in vitro assay disclosed herein.

The term “receptor” denotes a polypeptide capable of binding at leastone ligand. In one embodiment the receptor is a cell-surface receptorhaving an extracellular ligand-binding domain and, optionally, otherdomains (e.g. transmembrane domain, intracellular domain and/or membraneanchor). The receptor to be evaluated in the assay described herein maybe an intact receptor or a fragment or derivative thereof (e.g. a fusionprotein comprising the binding domain of the receptor fused to one ormore heterologous polypeptides). Moreover, the receptor to be evaluatedfor its binding properties may be present in a cell or isolated andoptionally coated on an assay plate or some other solid phase.

As used herein, “treatment” (and grammatical variations thereof such as“treat” or “treating”) refers to clinical intervention in an attempt toalter the natural course of the individual being treated, and can beperformed either for prophylaxis or during the course of clinicalpathology. Desirable effects of treatment include, but are not limitedto, preventing occurrence or recurrence of disease, alleviation ofsymptoms, diminishment of any direct or indirect pathologicalconsequences of the disease, preventing metastasis, decreasing the rateof disease progression, amelioration or palliation of the disease state,and remission or improved prognosis. In some embodiments, antibodies ofthe invention are used to delay development of a disease or to slow theprogression of a disease.

II. Tailor-Made Multispecific Binding Molecules

In most cell based diseases the targeting of the disease-related cellsvia antibody based binding of receptor molecules is one promisingapproach. However, the expression level of clinically relevant surfacereceptors (=target) varies from patient to patient and efficacy ofstandardized antibody based drugs is thus very different. This appliesspecifically for bi- and multispecific binding molecules whose mode ofaction is to target two different epitopes/receptors simultaneously.

One promising approach is to design a drug (here a bi- or multispecificbinding molecule) specifically for the particular/individual situationof the respective patient.

Each cell from an individual is different in view of the expressed cellsurface molecules, such as receptors, in number and kind This isespecially true for cancer cells and non-cancer cells. Thus, a cell canbe characterized by the cell surface molecules presented.

Based on expression profile data of clinically relevant surfacereceptors on disease-associated cells of a patient a series of bindingentities (for example Fab fragments) are specifically chosen from alibrary and combined to a multispecific binding molecule as the patientspecific drug. These selected binding molecules are specifically chosenwith respect to the respective disease-associated cell such as e.g. atumor cell based e.g. on the expression level of surface receptors and,thus, the need and phenotype of the individual patient.

Such a characterization can be effected by in vitro and in vivo basedcell imaging techniques. In vivo imaging techniques include e.g. opticalimaging, molecular imaging, fluorescence imaging, bioluminescenceimaging, MRI, PET, SPECT, CT, and intravital microscopy. In vitroimaging techniques include e.g. immunohistochemical staining of patientcells with e.g. fluorescently labeled antibodies recognizing specificcell surface markers and analysis of the fluorescence signals bymicroscopy. Alternatively the genotype/phenotype of the cells can beanalyzed after staining with labeled therapeutic or diagnosticantibodies using FACS-based methods.

In one embodiment the genotype/phenotype of patient-derived cells isdetermined by a FACS-based method. In one embodiment the cell surfacemarkers are determined by using fluorescently labeled diagnostic ortherapeutic antibodies. In one embodiment fluorescently labeledtherapeutic antibodies are used.

Certain diseases can be correlated with a change in the number ofspecific cell surface molecules or with occurrence of a new cell surfacemolecule.

Individuals affected by such a disease will display within certainranges a disease and/or an individual-specific cell surface markerpattern.

This has to be taken into consideration in order to provide to such anindividual a tailor-made, targeted therapeutic.

A number of therapeutic antibodies directed against cell surfacemolecules and their ligands are known which can be used for theselection and construction of tailor-made multi-specific targetingentities, such as Rituxan/MabThera/Rituximab, 2H7/Ocrelizumab,Zevalin/Ibrizumomab, Arzerra/Ofatumumab (CD20), HLL2/Epratuzumab,Inotuzomab (CD22), Zenapax/Daclizumab, Simulect/Basiliximab (CD25),Herceptin/Trastuzumab, Pertuzumab (Her2/ERBB2), Mylotarg/Gemtuzumab(CD33), Raptiva/Efalizumab (Cd11a),

Erbitux/Cetuximab (EGFR, epidermal growth factor receptor), IMC-1121B(VEGF receptor 2), Tysabri/Natalizumab (α4-subunit of α4β1 and α4β7integrins), ReoPro/Abciximab (gpIIb-gpIIa and αvβ3-integrin), OrthocloneOKT3/Muromonab-CD3 (CD3), Benlysta/Belimumab (BAFF), Tolerx/Oteliximab(CD3), Soliris/Eculizumab (C5 complement protein), Actemra/Tocilizumab(IL-6R), Panorex/Edrecolomab (EpCAM, epithelial cell adhesion molecule),CEA-CAM5/Labetuzumab (CD66/CEA, carcinoembryonic antigen), CT-11 (PD-1,programmed death-1 T-cell inhibitory receptor, CD-d279), H224G11 (c-Metreceptor), SAR3419 (C D19), IMC-A12/Cixutumumab (IGF-1R, insulin-likegrowth factor 1 receptor), MEDI-575 (PDGF-R, platelet-derived growthfactor receptor), CP-675, 206/Tremelimumab (cytotoxic T lymphocyteantigen 4), RO5323441 (placenta growth factor or PGF),HGS1012/Mapatumumab (TRAIL-R1), SGN-70 (CD70),Vedotin(SGN-35)/Brentuximab (CD30), and ARH460-16-2 (CD44).

For the determination of the cell surface markers present in a sample ofe.g. a patient, different methods are known. One exemplary method isbased on fluorescence activated cell sorting (FACS), in particular, theanalysis of specifically stained and sorted cell populations. In thismethod the phenotyping of the sample (cell population) is achieved byanalyzing individual cells with respect to the presented cell surfacemarkers using fluorescently labeled antibodies directed against thesemarkers optionally including the statistical distribution of surfacemarkers in the cell population. It is especially suitable to usetherapeutic antibodies that have been labeled with a fluorescent labelfor this purpose as therewith it is ensured that the later tailor-mademultispecific binding molecule will bind to the same epitope as thediagnostic antibody. The multispecific binding molecules/bispecificantibodies as reported herein can be used in the preparation ofmedicaments for the treatment of e.g. an oncologic disease, acardiovascular disease, an infectious disease, an inflammatory disease,an autoimmune disease, a metabolic (e.g., endocrine) disease, or aneurological (e.g. neurodegenerative) disease. Exemplary non-limitingexamples of these diseases are Alzheimer's disease, non-Hodgkin'slymphomas, B-cell acute and chronic lymphoid leukemias, Burkittlymphoma, Hodgkin's lymphoma, hairy cell leukemia, acute and chronicmyeloid leukemias, T-cell lymphomas and leukemias, multiple myeloma,glioma, Waldenstrom's macroglobulinemia, carcinomas (such as carcinomasof the oral cavity, gastrointestinal tract, colon, stomach, pulmonarytract, lung, breast, ovary, prostate, uterus, endometrium, cervix,urinary bladder, pancreas, bone, liver, gall bladder, kidney, skin, andtestes), melanomas, sarcomas, gliomas, and skin cancers, acuteidiopathic thrombocytopenic purpura, chronic idiopathic thrombocytopenicpurpura, dermatomyositis, Sydenham's chorea, myasthenia gravis, systemiclupus erythematosus, lupus nephritis, rheumatic fever, polyglandularsyndromes, bullous pemphigoid, diabetes mellitus, Henoch-Schonleinpurpura, post-streptococcal nephritis, erythema nodosum, Takayasu'sarteritis, Addison's disease, rheumatoid arthritis, multiple sclerosis,sarcoidosis, ulcerative colitis, erythema multiforme, IgA nephropathy,polyarteritis nodosa, ankylosing spondylitis, Goodpasture's syndrome,thromboangitis obliterans, Sjogren's syndrome, primary biliarycirrhosis, Hashimoto's thyroiditis, thyrotoxicosis, scleroderma, chronicactive hepatitis, polymyositis/dermatomyositis, polychondritis,pemphigus vulgaris, Wegener's granulomatosis, membranous nephropathy,amyotrophic lateral sclerosis, tabes dorsalis, giant cellarteritis/polymyalgia, pernicious anemia, rapidly progressiveglomerulonephritis, psoriasis, or fibrosing alveolitis.

A number of cell surface markers and their ligands are known. Forexample cancer cells have been reported to express at least one of thefollowing cell surface markers and or ligands, including but not limitedto, carbonic anhydrase IX, alpha-fetoprotein, alpha-actinin-4, A3(antigen specific for A33 antibody), ART-4, B7, Ba-733, BAGE,BrE3-antigen, CA125, CAMEL, CAP-1, CASP-8/m, CCCL19, CCCL21, CD1, CD1a,CD2, CD3, CD4, CDS, CD8, CD1-1A, CD14, CD15, CD16, CD18, CD19, CD20,CD21, CD22, CD23, CD25, CD29, CD30, CD32b, CD33, CD37, CD38, CD40,CD40L, CD45, CD46, CD54, CD55, CD59, CD64, CD66a-e, CD67, CD70, CD74,CD79a, CD80, CD83, CD95, CD126, CD133, CD138, CD147, CD154, CDC27,CDK-4/m, CDKN2A, CXCR4, CXCR7, CXCL12, HIF-1-alpha, colon-specificantigen-p (CSAp), CEA (CEACAM5), CEACAM6, c-met, DAM, EGFR, EGFRvIII,EGP-1, EGP-2, ELF2-M, Ep-CAM, Flt-1, Flt-3, folate receptor, G250antigen, GAGE, GROB, HLA-DR, HM1.24, human chorionic gonadotropin (HCG)and its subunits, HER2/neu, HMGB-1, hypoxia inducible factor (HIF-1),HSP70-2M, HST-2or 1a, IGF-1R, IFN-gamma, IFN-alpha, IFN-beta, IL-2,IL-4R, IL-6R, IL-13R, IL-15R, IL-17R, IL-18R, IL-6, IL-8, IL-12, IL-15,IL-17, IL-18, IL-25, insulin-like growth factor-1 (IGF-1), KC4-antigen,KS-1-antigen, KS 1-4, Le-Y, LDR/FUT, macrophage migration inhibitoryfactor (MIF), MAGE, MAGE-3, MART-1, MART-2, NY-ESO-1, TRAG-3, mCRP,MCP-1, MIP-1A, MIP-1B, MIF, MUC1, MUC2, MUC3, MUC4, MUC5, MUM-1/2,MUM-3, NCA66, NCA95, NCA90, pancreatic cancer mucin, placental growthfactor, p53, PLAGL2, prostatic acid phosphatase, PSA, PRAME, PSMA, P1GF,ILGF, ILGF-1R, IL-6, IL-25, RS5, RANTES, T101, SAGE, S100, survivin,survivin-2B, TAC, TAG-72, tenascin, TRAIL receptors, TNF-alpha,Tn-antigen, Thomson-Friedenreich antigens, tumor necrosis antigens,VEGFR, ED-B fibronectin, WT-1, 17-1A-antigen, complement factors C3,C3a, C3b, C5a, C5, an angiogenesis marker, bcl-2, bcl-6, Kras, cMET, anoncogene marker and an oncogene product (see, e.g., Sensi, et al., Clin.Cancer Res. 12 (2006) 5023-5032; Parmiani, et al, J. Immunol. 178 (2007)1975-1979; Novellino, et al., Cancer Immunol. Immunother. 54 (2005)187-207).

Thus, antibodies recognizing specific cell surface receptors includingtheir ligands can be used for specific and selective targeting andbinding to a number/multitude of cell surface markers that areassociated with a disease. A cell surface marker is a polypeptidelocated on the surface of a cell (e.g. a disease-related cell) that ise.g. associated with signaling event or ligand binding.

In one embodiment, for the treatment of cancer/tumors multispecificbinding molecules/bispecific antibodies are used that targettumor-associated antigens, such as those reported in Herberman,“Immunodiagnosis of Cancer”, in Fleisher (ed.), “The ClinicalBiochemistry of Cancer”, page 347 (American Association of ClinicalChemists (1979)) and in U.S. Pat. No. 4,150,149; U.S. Pat. No.4,361,544; and U.S. Pat. No. 4,444,744.

Reports on tumor associated antigens (TAAs) include Mizukami, et al.,(Nature Med. 11 (2005) 992-997); Hatfield, et al., (Curr. Cancer DrugTargets 5 (2005) 229-248); Vallbohmer, et al., (J Clin. Oncol. 23 (2005)3536-3544); and Ren, et al., (Ann. Surg. 242 (2005) 55-63), eachincorporated herein by reference with respect to the TAAs identified.

Where the disease involves a lymphoma, leukemia or autoimmune disorder,targeted antigens may be selected from the group consisting of CD4, CD5,CD8, CD14, CD15, CD19, CD20, CD21, CD22, CD23, CD25, CD33, CD37, CD38,CD40, CD40L, CD46, CD54, CD67, CD74, CD79a, CD80, CD126, CD138, CD154,CXCR4, B7, MUC1 or 1a, HM1.24, HLA-DR, tenascin, VEGF, P1GF, ED-Bfibronectin, an oncogene, an oncogene product (e.g., c-met or PLAGL2),CD66a-d, necrosis antigens, IL-2, T101, TAG, IL-6, MIF, TRAIL-R1 (DR4)and TRAIL-R2 (DR5).

A number of bispecific antibodies are known directed against twodifferent targets, such as BCMA/CD3, different antigens of the HERfamily in combination (EGFR, HER2, HER3), CD19/CD3, IL17RA/IL7R,IL-6/IL-23, IL-1-beta/IL-8, IL-6 or IL-6R/ IL-21 or IL-21R, firstspecificity directed to a glycoepitope of an antigen selected from thegroup consisting of Lewis x-, Lewis b- and Lewis y-structures, GloboH-structures, KH1, Tn-antigen, TF-antigen and carbohydrate structures ofMucins, CD44, glycolipids and glycosphingolipids, such as Gg3, Gb3, GD3,GD2, Gb5, Gm1, Gm2, sialyltetraosylceramide and a second specificitydirected to an ErbB receptor tyrosine kinase selected from the groupconsisting of EGFR, HER2, HER3 and HER4, GD2 in combination with asecond antigen binding site is associated with an immunological cellchosen from the group consisting of T-lymphocytes NK cell,B-lymphocytes, dendritic cells, monocytes, macrophages, neutrophils,mesenchymal stem cells, neural stem cells, ANG2/VEGF, VEGF/PDGFR-beta,Vascular Endothelial Growth Factor (VEGF) acceptor 2/CD3, PSMA/CD3,EPCAM/CD3, combinations of an antigen is selected from a groupconsisting of VEGFR-1, VEGFR-2, VEGFR-3, FLT3, c-FMS/CSF1R, RET, c-Met,EGFR, Her2/neu, HER3, HER4, IGFR, PDGFR, c-KIT, BCR, integrin and MMPswith a water-soluble ligand is selected from the group consisting ofVEGF, EGF, PIGF, PDGF, HGF, and angiopoietin, ERBB-3/C-MET,ERBB-2/C-MET, EGF receptor 1/CD3, EGFR/HER3, PSCA/CD3, C-MET/CD3,ENDOSIALIN/CD3, EPCAM/CD3, IGF-1R/CD3, FAPALPHA/CD3, EGFR/IGF-1R, IL17A/F, EGF receptor 1/CD3, and CD19/CD16.

Thus, it has been found that by using a modular approach as reportedherein tailor-made bispecific therapeutic antibodies can be provided.These antibodies are tailor-made with respect to cell surface moleculesactually present on the cells of an individual in need of a treatment orwith respect to ligands interacting with such a cell surface molecule.By determining the cell surface molecule status of an individual atailor-made combination of therapeutic targets can be chosen.

With this tailor-made generation of bispecific therapeutics by combining2 single therapeutic molecules for simultaneous targeting and binding totwo different epitopes an additive/synergistic effect can be expected incomparison to the single therapeutic molecules.

By using already available monospecific therapeutic binding entities,such as those derived from therapeutic antibodies, a fast and easyproduction of the required multispecific binding molecule can beachieved.

These avidity engineered binding molecules/antibodies can bind to two ormore cell surface markers present on a single cell. This binding is onlyavid if all/both binding entities simultaneously bind to the cell. Forthis purpose medium to low affine antibodies are especially suited. Thisallows also on the other hand to exclude less specific combinations ofbinding specificities during a screening process.

Selected patient specific multispecific binding molecules can be testedin various cellular in vitro assays/cell samples for relevant criteria(for example optimal binding/binding partners, optimal linker lengthetc.):

determining the phosphorylation status of phospho tyrosine kinases

determining c-Jun N-terminal kinase (JNK) inhibition

determining molecule induced apoptosis

binding assay performed with monospecific vs. multispecific bindingmolecule

determining of proliferation inhibition

With such an approach the generation of tailor-made and, thus, highlyefficient therapeutic molecules is possible. These molecules will havereduced side effects by improved targeting/delivery (e.g. payload fortumor cells) and improved targeting to target cell is based on higherselectivity and specificity of targeting component (comprising at leasttwo binding molecules).

The higher selectivity and specificity of multispecific binding moleculeis due to simultaneous binding (avidity) by the combination of two “lowaffinity” binders, which reduces possible “off-target” bindings.

Methods as Reported Herein

One aspect as reported herein is a method for producing a bispecificantibody comprising the step of incubating

-   -   (i) an antibody Fab fragment or a scFv antibody comprising        within the 20 C-terminal amino acid residues the amino acid        sequence LPX1TG (SEQ ID NO: 01, wherein X1 can be any amino acid        residue),    -   (ii) an antibody fragment comprising a full length antibody        heavy chain, a full length antibody light chain, and an antibody        heavy chain Fc-region polypeptide,    -   whereby the full length antibody heavy chain and the full length        antibody light chain are cognate antibody chains and the pair of        variable domains (VH and VL) thereof forms an antigen binding        site,    -   whereby the full length antibody heavy chain and the antibody        heavy chain

Fc-region polypeptide are covalently linked to each other via one ormore disulfide bonds forming an antibody hinge region, and

-   -   whereby the antibody heavy chain Fc-region has an oligoglycine        amino acid sequence at its N-terminus,    -   and    -   (iii) a Sortase A enzyme    -   and thereby producing the bispecific antibody.

One aspect as reported herein is a method for producing a bispecificantibody comprising the following steps

-   -   (i) determining surface makers present on the surface of a cell        in a sample and selecting thereof a first surface marker and a        second surface marker,    -   (ii) incubating (a) an antibody Fab fragment or a scFv antibody        fragment comprising within the 20 C-terminal amino acid residues        the amino acid sequence LPX1TG (SEQ ID NO: 01, wherein X1 can be        any amino acid residue), whereby the Fab fragment or scFv        specifically binds to the first surface marker, (b) an antibody        fragment comprising a full length antibody heavy chain, a full        length antibody light chain, and an antibody heavy chain        Fc-region polypeptide, whereby the full length antibody heavy        chain and the full length antibody light chain are cognate        antibody chains and the pair of variable domains (VH and VL)        thereof forms an antigen binding site that specifically binds to        the second surface marker, whereby the full length antibody        heavy chain and the antibody heavy chain Fc-region polypeptide        are covalently linked to each other via one or more disulfide        bonds forming an antibody hinge region, and whereby the antibody        heavy chain Fc-region has an oligoglycine amino acid sequence at        its N-terminus, and (c) a Sortase A enzyme    -   and thereby producing the bispecific antibody.

One aspect as reported herein is a method for determining a combinationof antigen binding sites comprising the following steps

-   -   (i) determining the binding specificity and/or affinity and/or        effector function and/or in vivo half-life of a multitude of        bispecific antibodies prepared by combining each member of a        first multitude of antibody Fab fragments or scFv antibody        fragments with each member of a second multitude of antibody        fragments comprising a full length antibody heavy chain, a full        length antibody light chain, and an antibody heavy chain        Fc-region polypeptide,    -   whereby the first multitude specifically binds to a first cell        surface molecule and the second multitude specifically binds to        a second cell surface molecule,    -   and    -   (ii) choosing the bispecific antibody with suitable binding        specificity and/or affinity and/or effector function and/or in        vivo half-life and thereby determining a combination of antigen        binding sites.

In one embodiment the combining is characterized by incubating theantibody Fab fragment or a scFv antibody fragment and the antibodyfragment comprising a full length antibody heavy chain, a full lengthantibody light chain, and an antibody heavy chain Fc-region polypeptide,with a Sortase A enzyme.

In one embodiment the Fab fragment or scFv antibody fragment compriseswithin the 20 C-terminal amino acid residues the amino acid sequenceLPX1TG (SEQ ID NO: 01, wherein X1 can be any amino acid residue).

In one embodiment the full length antibody heavy chain and the fulllength antibody light chain of the one-armed antibody fragment arecognate antibody chains and the pair of variable domains (VH and VL)thereof forms an antigen binding site that specifically binds to thesecond surface marker, the full length antibody heavy chain and theantibody heavy chain Fc-region polypeptide are covalently linked to eachother via one or more disulfide bonds forming an antibody hinge region,and the antibody heavy chain Fc-region polypeptide has an oligoglycineamino acid sequence at its N-terminus.

In one embodiment of all aspects the antibody Fab fragment or the scFvantibody comprises within the 20 C-terminal amino acid residues theamino acid sequence G_(n)SLPX1TG (SEQ ID NO:02, wherein X1 can be anyamino acid residue, with n=1, 2 or 3).

In one embodiment of all aspects the antibody Fab fragment or the scFvantibody comprises within the 20 C-terminal amino acid residues theamino acid sequence GSLPX1TGGSGS (SEQ ID NO: 03, wherein X1 can be anyamino acid residue).

In one embodiment of all aspects the antibody Fab fragment or the scFvantibody comprises the amino acid sequence X2GSLPX1TGGSGS (SEQ ID NO:05, wherein X1 can be any amino acid residue, whereby X2 can be anyamino acid residue except G.

In one embodiment of all aspects the antibody Fab fragment or the scFvantibody comprises the amino acid sequence G_(n)SLPX1TGGSGSX3 (SEQ IDNO: 06, wherein X1 can be any amino acid residue, with n=1, 2 or 3)within the 20 C-terminal amino acid residues, whereby X3 is an aminoacid sequence tag.

In one embodiment of all aspects the antibody Fab fragment or the scFvantibody comprises the amino acid sequence X2GSLPX1TGGSGSX3 (SEQ ID NO:07, wherein X1 can be any amino acid residue) within the 20 C-terminalamino acid residues whereby X2 can be any amino acid residue except Gand X3 is an amino acid sequence tag.

In one embodiment of all aspects the antibody heavy chain Fc-regionpolypeptide comprises two glycine residues at its N-terminus.

In one embodiment of all aspects the one armed antibody Fc-regioncomprises the amino acid sequence GGCPX4C (SEQ ID NO: 08) at theN-terminus of its heavy chain Fc-region polypeptide, whereby X4 iseither S or P.

In one embodiment of all aspects X1 is E.

One aspect as reported herein is a multispecific bindingmolecule/bispecific antibody obtained by a method as reported herein.

One aspect is a multispecific binding molecule/bispecific antibodycomprising the amino acid sequence LPX1TG (SEQ ID NO: 01, wherein X1 canbe any amino acid residue) in one of its heavy chains.

In one embodiment the multispecific binding molecule/bispecific antibodycomprises the amino acid sequence G_(n)SLPX1TG (SEQ ID NO: 02, whereinX1 can be any amino acid residue, with n=1, 2 or 3) in one of its heavychains.

In one embodiment the multispecific binding molecule/bispecific antibodycomprises the amino acid sequence G.SLPX1TGGCPX4C (SEQ ID NO: 09,wherein X1 can be any amino acid residue, wherein X4 can be S or P, withn=1, 2 or 3) in one of its heavy chains.

In one embodiment the multispecific binding molecule/bispecific antibodycomprises the amino acid sequence X2GSLPX1TGGCPX4C (SEQ ID NO: 10,wherein X1 can be any amino acid residue, wherein X4 can be S or P) inone of its heavy chains, whereby X2 can be any amino acid residue exceptG.

In one embodiment X1 is E.

One aspect as reported herein is a pharmaceutical formulation comprisingan antibody/multispecific binding molecule as reported herein.

One aspect as reported herein is the use of a bispecificantibody/multispecific binding molecule as reported herein in themanufacture of a medicament.

In one embodiment the medicament is for the treatment of cancer.

One aspect as reported herein is a method of treating an individualhaving cancer comprising administering to the individual an effectiveamount of a bispecific antibody/multispecific binding molecule asreported herein.

One aspect as reported herein is a method for destroying cancer cells inan individual comprising administering to the individual an effectiveamount of a bispecific antibody/multispecific binding molecule asreported herein.

In one embodiment of all aspects as reported herein the Fc-region is ahuman Fc-region, or a variant thereof.

In one embodiment the human Fc-region is of the human IgG1 subclass, orof the human IgG2 subclass, or of the human IgG3 subclass, or of thehuman IgG4 subclass. In one embodiment the Fc-region is a humanFc-region of the human IgG1 subclass or of the human IgG4 subclass.

In one embodiment the human Fc-region comprises a mutation of thenaturally occurring amino acid residue at least at one of the followingamino acid positions 228, 233, 234, 235, 236, 237, 297, 318, 320, 322,329, and/or 331 to a different residue, wherein the residues in theFc-region are numbered according to the EU index of Kabat.

In one embodiment the human Fc-region comprises a mutation of thenaturally occurring amino acid residue at position 329 and at least onefurther mutation of at least one amino acid selected from the groupcomprising amino acid residues at position 228, 233, 234, 235, 236, 237,297, 318, 320, 322 and 331 to a different residue, wherein the residuesin the Fc-region are numbered according to the EU index of Kabat. Thechange of these specific amino acid residues results in an altering ofthe effector function of the Fc-region compared to the non-modified(wild-type) Fc-region.

In one embodiment the human Fc-region has a reduced affinity to thehuman FcγRIIIA and/or FcγRIIA and/or FcγRI compared to a conjugatecomprising the corresponding wild-type IgG Fc-region.

In one embodiment the amino acid residue at position 329 in the humanFc-region is substituted with glycine, or arginine, or an amino acidresidue large enough to destroy the proline sandwich within theFc-region.

In one embodiment the mutation of the naturally occurring amino acidresidue is S228P, E233P, L234A, L235A, L235E, N297A, N297D, P329G,and/or P331S. In one embodiment the mutation is L234A and L235A if theFc-region is of human IgG1 subclass or S228P and L235E if the Fc-regionis of human IgG4 subclass. In one embodiment the Fc-region comprises themutation P329G.

By the combination of two mutations at defined positions in theFc-region a complete reduction of the Fc-region associated effectorfunction can be achieved.

The selection of an effector function eliciting Fc-region is dependenton the intended use of the multispecific binding molecules/bispecificantibody.

If the desired use is the functional neutralization of a soluble targeta non-effector function eliciting subclass or variant should beselected.

If the desired use is the removal of a (soluble) target an effectorfunction eliciting subclass or variant should be selected.

If the desired use is the antagonization of a cell-bound target anon-effector function eliciting subclass or variant should be selected.

If the desired use is the removal of a target presenting cell aneffector function eliciting subclass or variant should be selected.

The circulating half-life of an antibody or antibody Fc-region conjugatecan be influenced by modulating the Fc-region-FcRn interaction.

The minimization or even removal of antibody-dependent cell-mediatedcytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) can beachieved by so called hinge-region amino acid changes/substitutions.

The minimization or even removal of the activation of the classicalcomplement cascade can be achieved by so called hinge-region amino acidchanges/substitutions.

An increase of the circulatory half-life of an antibody or antibodyFc-region conjugate can be achieved by increased binding to the neonatalFc receptor and results in an improved efficacy, a reduced dose orfrequency of administration, or an improved delivery to the target. Areduction of the circulatory half-life of an antibody or antibodyFc-region conjugate can be achieved by reduced binding to the neonatalFc receptor and results in a reduced whole body exposure or an improvedtarget-to-non-target binding ratio.

Generally, the method as reported herein is applicable to the productionof antibody Fc-region conjugates comprising either a wild-type Fc-regionor an altered/variant Fc-region.

In one embodiment the Fc-region is a human Fc-region.

In one embodiment the Fc-region is “conceptual” and, while it does notphysically exist, the antibody engineer may decide upon a variantFc-region to be used.

In one embodiment the nucleic acid encoding the Fc-region part of theantibody Fc-region conjugate is altered to generate a variant nucleicacid sequence encoding the variant Fc-region part of the antibodyFc-region conjugate.

The nucleic acid encoding the amino acid sequence of the Fc-region partof the antibody Fc-region conjugate can be prepared by a variety ofmethods known in the art. These methods include, but are not limited to,preparation by site-directed (or oligonucleotide-mediated) mutagenesis,PCR mutagenesis, and cassette mutagenesis of an earlier prepared DNAencoding the polypeptides of the antibody Fc-region conjugate.

The Fc-region interacts with a number of receptors or ligands includingbut not limited to Fc receptors (e.g. FcγRI, FcγRIIA, FcγRIIIA), thecomplement protein C1q, and other molecules such as proteins A and G.These interactions are essential for a variety of effector functions anddownstream signaling events including, but not limited to, antibodydependent cell-mediated cytotoxicity (ADCC), antibody dependent cellularphagocytosis (ADCP) and complement dependent cytotoxicity (CDC).

In one embodiment the antibody Fc-region conjugate (as produced with themethod as reported herein) has at least one or more of the followingproperties: reduced or ablated effector function (ADCC and/or CDC and/orADCP), reduced or ablated binding to Fc receptors, reduced or ablatedbinding to C1q, or reduced or ablated toxicity.

In one embodiment the antibody Fc-region conjugate (as produced with themethod as reported herein) comprises a wild-type Fc-region that has atleast two amino acid mutations, additions, or deletions.

In one embodiment the antibody Fc-region conjugate (as produced with themethod as reported herein) has a reduced affinity to a human Fc receptor(FcγR) and/or a human complement receptor compared to an antibody orantibody Fc-region conjugate comprising a wild-type human Fc-region.

In one embodiment the antibody Fc-region conjugate (as produced with themethod as reported herein) comprises an Fc-region that has a reducedaffinity to a human Fc receptor (FcγR) and/or human complement receptorcompared to an antibody or antibody Fc-region conjugate comprising awild-type human Fc-region.

In one embodiment the antibody Fc-region conjugate (as produced with themethod as reported herein) has reduced affinity to at least one ofFcγRI, FcγRII, and/or FcγRIIIA. In one embodiment the affinity to FcγRIand FcγRIIIA is reduced. In one embodiment the affinity to FcγRI, FcγRIIand FcγRIIIA is reduced.

In one embodiment the affinity to FcγRI, FcγRIIIA and C1q is reduced.

In one embodiment the affinity to FcγRI, FcγRII, FcγRIIIA and C1q isreduced.

In one embodiment the antibody Fc-region conjugate (as produced with themethod as reported herein) has a reduced ADCC compared to an antibody orantibody Fc conjugate comprising a wild-type Fc-region. In oneembodiment the ADCC is reduced by at least 20% compared to the ADCCinduced by an Fc-region fusion polypeptide or conjugate comprising awild-type Fc-region.

In one embodiment the antibody Fc-region conjugate (as produced with themethod as reported herein) has an ADCC and CDC induced by the Fc-regionthat is decreased or ablated compared to an antibody Fc-region conjugatecomprising a wild-type Fc-region.

In one embodiment the antibody Fc-region conjugate (as produced with themethod as reported herein) has a decreased ADCC, CDC, and ADCP comparedto an OA-Fc-region conjugate comprising a wild-type Fc-region.

In one embodiment the antibody Fc-region conjugate comprises at leastone amino acid substitution in the Fc-region that is selected from thegroup comprising S228P, E233P, L234A, L235A, L235E, N297A, N297D, P329G,and P331S.

In one embodiment the wild-type Fc-region is a human IgG1 Fc-region or ahuman IgG4 Fc-region.

In one embodiment the antibody Fc-region comprises besides a mutation ofthe amino acid residue proline at position 329 at least one furtheraddition, mutation, or deletion of an amino acid residue in theFc-region that is correlated with increased stability of the antibodyFc-region conjugate.

In one embodiment the further addition, mutation, or deletion of anamino acid residue in the Fc-region is at position 228 and/or 235 of theFc-region if the Fc-region is of IgG4 subclass. In one embodiment theamino acid residue serine at position 228 and/or the amino acid residueleucine at position 235 is/are substituted by another amino acid. In oneembodiment the antibody Fc-region conjugate comprises a proline residueat position 228 (mutation of the serine residue to a proline residue).In one embodiment the antibody Fc-region conjugate comprises a glutamicacid residue at position 235 (mutation of the leucine residue to aglutamic acid residue).

In one embodiment the Fc-region comprises three amino acid mutations. Inone embodiment the three amino acid mutations are P329G, S228P and L235Emutation (P329G/SPLE).

In one embodiment the further addition, mutation, or deletion of anamino acid residue in the Fc-region is at position 234 and/or 235 of theFc-region if the Fc-region is of IgG1 subclass. In one embodiment theamino acid residue leucine at position 234 and/or the amino acid residueleucine at position 235 is/are mutated to another amino acid.

In one embodiment the Fc-region comprises an amino acid mutation atposition 234, wherein the leucine amino acid residue is mutated to analanine amino acid residue.

In one embodiment the Fc-region comprises an amino acid mutation atposition 235, wherein the leucine amino acid residue is mutated to analanine amino acid residue.

In one embodiment the Fc-region comprises an amino acid mutation atposition 329, wherein the proline amino acid residue is mutated to aglycine amino acid residue, an amino acid mutation at position 234,wherein the leucine amino acid residue is mutated to an alanine aminoacid residue, and an amino acid mutation at position 235, wherein theleucine amino acid residue is mutated to an alanine amino acid residue.

Fc-region variants with increased affinity for FcRn have longer serumhalf-lives, and such molecules will have useful applications in methodsof treating mammals where long systemic half-life of the administeredantibody Fc-region conjugate is desired, e.g., to treat a chronicdisease or disorder.

Antibody Fc-region conjugates with decreased FcRn binding affinity haveshorter serum half-lives, and such molecules will have usefulapplications in methods of treating mammals where a shorter systemichalf-life of the administered antibody Fc-region conjugate is desired,e.g. to avoid toxic side effects or for in vivo diagnostic imagingapplications. Fc-region fusion polypeptides or conjugates with decreasedFcRn binding affinity are less likely to cross the placenta, and thusmay be utilized in the treatment of diseases or disorders in pregnantwomen.

Fc-regions with altered binding affinity for FcRn is in one embodimentan Fc-region with an amino acid alteration at one or more of the aminoacid positions 238, 252, 253, 254, 255, 256, 265, 272, 286, 288, 303,305, 307, 309, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382,386, 388, 400, 413, 415, 424, 433, 434, 435, 436, 439, and/or 447.

The Fc-region is in one embodiment an Fc-region with one or more aminoacid alterations at the amino acid positions 252, 253, 254, 255, 288,309, 386, 388, 400, 415, 433, 435, 436, 439, and/or 447.

Fc-regions which display increased binding to FcRn comprise in oneembodiment one or more amino acid alterations at the amino acidpositions 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340,356, 360, 362, 376, 378, 380, 382, 413, 424, and/or 434.

In one embodiment the Fc-region is an Fc-region of the IgG1 subclass andcomprises the amino acid mutations P329G, and/or L234A and L235A.

In one embodiment the Fc-region is an Fc-region of the IgG4 subclass andcomprises the amino acid mutations P329G, and/or S228P and L235E.

In one embodiment the antibody Fc-region comprises the mutation T366W inthe first heavy chain Fc-region polypeptide and the mutations T366S,L368A and Y407V in the second heavy chain Fc-region polypeptide, whereinthe numbering is according to the EU index of Kabat.

In one embodiment the antibody Fc-region comprises the mutation S354C inthe first heavy chain Fc-region polypeptide and the mutation Y349C inthe second heavy chain Fc-region polypeptide.

Enzymatic Conjugation using Sortase A

A bispecific antibody comprising a one-armed antibody (OA-Fc) and one ormore antigen binding domains can be obtained by using the enzyme SortaseA.

Many gram-positive bacteria use sortase to covalently anchor a varietyof surface proteins including virulence factors to their cell wall(peptidoglycan). Sortases are extracellular membrane associated enzymes.The wild-type Staphylococcus aureus Sortase A (SrtA) is a polypeptide of206 amino acids with an N-terminal membrane-spanning region. In a firststep, sortase A recognizes substrate proteins that contain a LPX1TGamino acid sequence motif and cleaves the amide bond between the Thr andGly by means of an active-site Cys. This peptide cleaving reactionresults in a sortase A thioester intermediate. In a second step thethioester acyl-enzyme intermediate is resolved by nucleophilic attack ofan amino group of oligoglycine containing second substrate polypeptide(corresponding to the pentaglycine unit of peptidoglycan in S. aureus)leading to a covalently linked cell wall protein and the regeneration ofsortase A. In the absence of oligoglycine nucleophiles, the acyl-enzymeintermediate is hydrolyzed by a water molecule.

Sortase-mediated ligation/conjugation has begun to be applied for avariety of protein engineering and bioconjugation purposes. This newtechnique enables the introduction of natural and unnaturalfunctionalities into LPX1TG-tagged recombinant or chemically synthesizedpolypeptides. Examples include the covalent attachment of oligoglycinederivatized polymers (e.g. PEG), fluorophores, vitamins (e.g. biotin andfolate) lipids, carbohydrates, nucleic acids, synthetic peptides andproteins (e.g. GFP) (Tsukiji, S. and Nagamune, T., ChemBioChem 10 (2009)787-798; Popp, M. W.-L. and Ploegh, H. L., Angew. Chem. Int. Ed. 50(2011) 5024-5032).

It has been shown that a triglycine and even a diglycine motif of theamino component is sufficient for the SrtA-mediated ligation step(Clancy, K. W., et al., Peptide Science 94 (2010) 385-396).

For the enzymatic conjugation a soluble truncated sortase A lacking themembrane-spanning region (SrtA; amino acid residues 60-206 ofStaphylococcus aureus SrtA) can be used (Ton-That, H., et al., Proc.Natl. Acad. Sci. USA 96 (1999) 12424-12429; Ilangovan, H., et al., Proc.Natl. Acad. Sci. USA 98 (2001) 6056-6061). The truncated soluble sortaseA variant can be produced in E.coli.

An antibody Fc-region comprising an oligoglycine at least at one of itsN-termini (G_(m), m=2, or 3, or 4, or 5) can be expressed and purifiedfrom the supernatant of eukaryotic cells (e.g. HEK293 cells, CHO cells).

A binding entity (e.g. a single chain antigen binding polypeptide suchas a scFv, a scFab, or a darpin, or a multi chain antigen bindingpolypeptide such as a dsFv or a Fab) comprising the SrtA recognitionmotif at the C-terminus of one polypeptide chain can be expressed andpurified from the supernatant of eukaryotic cells (e.g. HEK293 cells,CHO cells).

One aspect as reported herein is an bispecific antibody that is obtainedby conjugating an antigen binding polypeptide/domain (e.g. scFv or Fab)to an one-armed antibody variant (OA-Fc) using the enzyme Sortase A,wherein a sortase recognition sequence is located at the C-terminus ofthe single chain antigen binding polypeptide (e.g. scFv, scFab ordarpin) or the C-terminus of one polypeptide chain of the multi chainantigen binding complex (e.g. dsFv or Fab), and wherein a double ortriple glycine motif is located at the N-terminus of the Fc-chain of theone-armed antibody variant (OA-Fc-Gm; m=2 or 3). An one-armed antibodyFab or scFv conjugate comprising an antibody Fab fragment (OA-Fc˜Fab) ora scFv antibody fragment (OA-Fc˜scFv) and an one-armed antibody (OA-Fc)can be obtained in high yield in an enzymatic conjugation by using (i) apolypeptide comprising the amino acid sequence G_(n)SLPX1TG (SEQ IDNO:02, wherein X1 can be any amino acid residue, with n=1, 2 or 3) inits C-terminal region, (ii) an heavy chain Fc-region polypeptidecomprising an oligoglycine at its N-terminus, and (iii) the enzymeSortase A.

With this combination of reagents

-   i) the reverse reaction recognizing the LPX1TG amino acid sequence    within the product conjugate as substrate, and/or-   ii) the generation of a dead-end hydrolysis polypeptide fragment    (polypeptide with without/cleaved LPX1TG recognition sequence    generated through cleavage of the thioacyl-binding entity Sortase A    intermediate by water instead by the G_(m)-antibody Fc-region    nucleophile)

that is normally occurring at increased reaction times can be reduced oreven eliminated.

Different combinations of C-terminal and N-terminal amino acid sequencecombinations have been tested.

In more detail, as an exemplary binding entity an antibody Fab fragmentwas used and as exemplary antibody Fc-region a one armed antibodyFc-region (OA-Fc-region=a pair of a full length antibody heavy chain andits cognate light chain and an heavy chain antibody Fc-regionpolypeptide) was used. Three different sequences at the C-terminus ofthe antibody Fab fragment VH-CH1 heavy chain and at the N-terminus ofthe OA-Fc-region respectively were conjugated using the exemplarytranspeptidase Sortase A. Nine different conjugates were obtained. Theprogress/efficiency of the coupling reaction was determined at differenttime points. To this end aliquots of the transpeptidation reactions wereanalyzed by SDS-PAGE. The efficiency of ligation was estimateddensitometrically from the gel. The results are given in the followingTable 1.

TABLE 1 One armed antibody Fc-region Fab VH-CH1 (OA-Fc-region) heavychain GGGDKTHTCPPC GGHTCPPC GGCPPC KSCGGGSLPETGGSGSHHHH approx. approx.approx. HH 54% 62% 73% KSCGSLPETGGSGSHHHHHH approx. approx. approx. 56%56% 73% KSCLPETGGSGSHHHHHH approx. approx. approx. 52% 54% 54%

In one embodiment the Fab antibody fragment or scFv antibody fragmentcomprises the amino acid sequence GSLPX1TGGSGS (SEQ ID NO: 03, whereinX1 can be any amino acid residue) within the 20 C-terminal amino acidresidues.

In one embodiment the Fab antibody fragment or scFv antibody fragmentcomprises the amino acid sequence X2GSLPX1TGGSGS (SEQ ID NO: 05, whereinX1 can be any amino acid residue, whereby X2 can be any amino acidresidue except G.

In one embodiment the Fab antibody fragment or scFv antibody fragmentcomprises the amino acid sequence G_(n)SLPX1TGGSGSX3 (SEQ ID NO: 06,wherein X1 can be any amino acid residue, with n=1, 2 or 3) within the20 C-terminal amino acid residues, whereby X3 is an amino acid sequencetag.

In one embodiment the Fab antibody fragment or scFv antibody fragmentcomprises the amino acid sequence X2GSLPX1TGGSGSX3 (SEQ ID NO: 07,wherein X1 can be any amino acid residue, with n=1, 2 or 3) within the20 C-terminal amino acid residues whereby X2 can be any amino acidresidue except G and X3 is an amino acid sequence tag.

The “Combimatrix” Approach

It is desirable to combine a first binding entity, such as an antibodyFab fragment, with another specific binding entity, such as a secondantibody Fab fragment or a one-armed antibody fragment comprising a fulllength heavy chain and its cognate light chain and a disulfide linkedheavy chain Fc-region polypeptide. In addition it is possible to screen,whether a first binding entity shows better properties when linking itto a number of different other binding entities. Using a so-calledCombimatrix approach, a multitude of combinations of binding entitiescan be addressed in an easy way. It has to be pointed out that thesecond binding entities can either bind to differenttargets/epitopes/antigens, or can bind to the same antigen but todifferent epitopes, or can bind to the same epitope but be differentvariants of a single binding entity (e.g. humanization candidates).

In this scenario, an automated platform can perform the tasks topipette, purify and combine the binding entities and their reactions orderivatives. Any platform that uses e.g. 96-well plates or other highthroughput formats is suitable, such as an Eppendorf epMotion 5075vacpipetting robot.

First, cloning of the binding entity encoding constructs is performed.The plasmids with the binding entity encoding nucleic acids are usuallyobtained by gene synthesis, whereby the C-terminal region of one encodedbinding entity contains a sortase-motive and a His-tag and oneN-terminal region of the respective other binding entity comprises onoligoglycine motif, or by cloning of the variable domains via B-cell PCRand sequence- and ligation-independent cloning (SLIC) into anappropriate vector containing necessary elements like the constantregion, a sortase motive and a His-tag respectively. The plasmids areindividually transferred into a separate well of a multi-well plate (awhole plate can be loaded). Thereafter, the plasmids are digested with arestriction enzyme mix that cuts out the binding entity-coding region.It is desirable to design all gene synthesis in a way that only onerestriction enzyme mix is needed for all plasmids. Afterwards, anoptional cleaning step yields purified DNA fragments. These fragmentsare ligated into a plasmid backbone that had been cut out of an acceptorvector with the same restriction mix as mentioned above. Alternatively,the cloning procedure can be performed by a SLIC-mediated cloning step(see e.g. PCT/EP2012/076155). After ligation, the automated platformstransfers all ligation mixes into a further multi-well plate withcompetent E. coli cells (e.g. Top10 Multi Shot, Invitrogen) and atransformation reaction is performed. The cells are cultivated to thedesired density. From an aliquot of the cultivation mixture glycerolstocks can be obtained. From the culture plasmid is isolated (e.g. usinga plasmid isolation mini kit (e.g. NucleoSpin 96 Plasmid, Macherey&Nagel)). Plasmid identity is checked by digesting an aliquot with anappropriate restriction mix and polyacrylamide gel electrophoresis (e.g.E-Gel 48, Invitrogen). Afterwards a new plate can be loaded with analiquot of the plasmid for performing a control sequencing reaction.

In the next step the binding entities are expressed. Therefore, HEKcells are seeded onto a multi-well plate (e.g. a 48-well-plate) or smallshaker flasks and are transfected with the isolated plasmids (containingthe binding entity-coding region in an appropriate backbone vector).Transfected HEK cells are cultivated for several days and harvested(e.g. by filtrating through a 1.2 μm and a 0.22 μm filter plate by usinga vacuum station). Titers can be monitored by performing e.g. an ELISA.

The binding entities can be linked to the each other using asortase-mediated transpeptidation reaction. The first binding entity,the second binding entity, and the sortase reaction mix can be combinedin a multi-well format. After incubation at 37° C. for 4-72 h (e.g. 16hours), the conjugates can be harvested by using a negative His-tagselection procedure (the mixture is applied onto e.g. His MultiTrap HPplates (GE Healthcare) and filtrated, whereby all molecules that stillhave a His-tag are bound on the chromatography column, whereas theconjugates are found in the filtrate; with the filtrate a bufferexchange should be made, e.g. by applying the conjugate onto anultrafiltration membrane or by using a plate containing an affinitymedium that is specific for one of the binding entities.

The multispecific binding molecules can be made using the Combimatrixapproach, see Table below).

1 2 3 4 5 6 7 8 9 10 11 A 1A 2A 3A 4A 5A 6A 7A 8A 9A 10A 11A B 1B ...... ... ... ... ... ... ... ... ... C 1C ... ... ... ... ... ... ... ...... ... D 1D ... ... ... ... ... ... ... ... ... ... E 1E  ... ... ...... ... ... ... ... ... ... F 1F  ... ... ... ... ... ... ... ... ...... G 1G ... ... ... ... ... ... ... ... 10G 11G

In the first row of a multi-well plate different first binding entitiescomprising a C-terminal Sortase motif of equal molar concentrations arepipetted into each well (excluding first well of the first row),designated in arabic numbers (e.g. 1 to 11). In the first column of thesame plate, different second binding entities comprising an oligoglycinein the N-terminal region of equal molar concentrations are pipetted intoeach well (excluding first well of the first column), designated inletters (e.g. A to G). Thereafter all first binding entities of thefirst row are combined with all second binding entities of the firstcolumn (e.g. resulting in 77 combinations in a 96-well plate),designated by a combination of number and letter (e.g. 1A to 11G). Toall combinations Sortase in an appropriate buffer is added. After theenzymatic conjugation has been performed, an optional purification stepcan be performed. The multispecific binding molecules are then ready forevaluation in cell-based assays.

III. Recombinant Methods

The ligation components of an OA-Fc-region conjugate, in particular, theone-armed antibody variant (OA-Fc-Gm) and the single chain antigenbinding polypeptide (e.g. scFv, scFab or darpin) or the multi chainantigen binding complex (e.g. dsFv or Fab) may be produced usingrecombinant methods and compositions, see e.g. U.S. Pat. No. 4,816,567.

In one aspect a method of making an OA-Fc˜polypeptide conjugate isprovided, wherein the method comprises (i) culturing a first host cellcomprising a nucleic acid encoding the one-armed antibody variant(OA-Fc-Gm) part of the conjugate under conditions suitable forexpression/secretion of the one-armed antibody variant (OA-Fc-Gm) andoptionally recovering the OA-Fc-Gm part from the host cell (or host cellculture medium) and (ii) culturing a second host cell comprising anucleic acid encoding the polypeptide part of the conjugate underconditions suitable for expression/secretion of the polypeptide andoptionally recovering the polypeptide part from the host cell (or hostcell culture medium) and (iii) conjugating the recombinantly producedparts of the OA-Fc˜polypeptide conjugate enzymatically using Sortase Amediated transpeptidation.

For recombinant production of the OA-Fc-Gm part of the OA-Fc˜polypeptideconjugate and the polypeptide part, a nucleic acid encoding the OA-Fc-Gmpart and the polypeptide part of the OA-Fc˜polypeptide conjugate, e.g.,as described above, is isolated and inserted into one or more vectorsfor further cloning and/or expression/secretion in a host cell. Suchnucleic acid may be readily isolated and/or produced using conventionalprocedures.

Suitable host cells for cloning or expression/secretion ofpolypeptide-encoding vectors include prokaryotic or eukaryotic cellsdescribed herein. For example, polypeptides may be produced in bacteria,in particular when glycosylation and Fc effector function are not needed(see, e.g., U.S. Pat. No. 5,648,237, U.S. Pat. No. 5,789,199, and U.S.Pat. No. 5,840,523, Charlton, Methods in Molecular Biology 248 (2003)245-254 (B.K.C. Lo, (ed.), Humana Press, Totowa, N.J.), describingexpression of antibody fragments in E. coli.). After expression, thepolypeptide may be isolated from the bacterial cell paste in a solublefraction or may be isolated from the insoluble fraction so calledinclusion bodies which can be solubilized and refolded to bioactiveforms. Thereafter the polypeptide can be further purified.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeasts are suitable cloning or expression hosts forpolypeptide-encoding vectors, including fungi and yeast strains whoseglycosylation pathways have been “humanized”, resulting in theproduction of a polypeptide with a partially or fully humanglycosylation pattern (see e.g. Gerngross, Nat. Biotech. 22 (2004)1409-1414, and Li, et al., Nat. Biotech. 24 (2006) 210-215).

Suitable host cells for the expression of glycosylated polypeptides arealso derived from multicellular organisms (invertebrates andvertebrates). Examples of invertebrate cells include plant and insectcells. Numerous baculoviral strains have been identified which may beused in conjunction with insect cells, particularly for transfection ofSpodoptera frugiperda cells.

Plant cell cultures can also be utilized as hosts (see, e.g., U.S. Pat.No. 5,959,177, U.S. Pat. No. 6,040,498, U.S. Pat. No. 6,420,548, U.S.Pat. No. 7,125,978, and U.S. Pat. No. 6,417,429 (describingPLANTIBODIES™ technology for producing antibodies in transgenicplants)).

Vertebrate cells may also be used as hosts. For example, mammalian celllines that are adapted to grow in suspension may be useful. Otherexamples of useful mammalian host cell lines are the COS-7 cell line(monkey kidney CV1 cell transformed by SV40; the HEK293 cell line (humanembryonic kidney) BHK cell line (baby hamster kidney); the TM4 mousesertoli cell line (TM4 cells as described, e.g., in Mather, Biol.Reprod. 23 (1980) 243-251); the CV1 cell line (monkey kidney cell); theVERO-76 cell line (African green monkey kidney cell); the HELA cell line(human cervical carcinoma cell); the MDCK cell line (canine kidneycell); the BRL-3A cell line (buffalo rat liver cell); the W138 cell line(human lung cell); the HepG2 cell line (human liver cell); the MMT060562 cell line (mouse mammary tumor cell); the TRI cell line, asdescribed, e.g., in Mather, et al., Annals N.Y. Acad. Sci. 383 (1982)44-68; the MRCS cell line; and FS4 cells-line. Other useful mammalianhost cell lines include the CHO cell line (Chinese hamster ovary cell),including DHFR negative CHO cell lines (Urlaub, et al., Proc. Natl.Acad. Sci. USA 77 (1980) 4216), and myeloma cell lines such as Y0, NS0and Sp2/0 cell line. For a review of certain mammalian host cell linessuitable for polypeptide production, see, e.g., Yazaki, and Wu, Methodsin Molecular Biology, Antibody Engineering 248 (2004) 255-268 (B.K.C.Lo, (ed.), Humana Press, Totowa, N.J.).

IV. Methods and Compositions for Diagnostics and Detection

In certain embodiments, any of the bispecific antibodies provided hereinis useful for detecting the presence of one or both antigens in abiological sample. The term “detecting” as used herein encompassesquantitative or qualitative detection. In certain embodiments, abiological sample comprises a cell or tissue, such as biopsies of cancercells.

In one embodiment, a bispecific antibody for use in a method ofdiagnosis or detection is provided. In a further aspect, a method ofdetecting the presence of cancer cells in a biological sample isprovided. In certain embodiments, the method comprises contacting thebiological sample with a bispecific antibody as described herein underconditions permissive for binding of the bispecific antibody to itsantigen or antigens, and detecting whether a complex is formed betweenthe bispecific antibody and its antigen or antigens. Such method may bean in vitro or in vivo method.

Exemplary disorders that may be diagnosed using an antibody of theinvention include cancer.

In certain embodiments, labeled bispecific antibodies are provided.Labels include, but are not limited to, labels or moieties that aredetected directly (such as fluorescent, chromophoric, electron-dense,chemiluminescent, and radioactive labels), as well as moieties, such asenzymes or ligands, that are detected indirectly, e.g., through anenzymatic reaction or molecular interaction. Exemplary labels include,but are not limited to, the radioisotopes ³²P, ¹⁴C, ¹²⁵I, ³H, and ¹³¹I,fluorophores such as rare earth chelates or fluorescein and itsderivatives, rhodamine and its derivatives, dansyl, umbelliferone,luceriferases, e.g., firefly luciferase and bacterial luciferase (U.S.Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones,horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase,glucoamylase, lysozyme, saccharide oxidases, e.g., glucose oxidase,galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclicoxidases such as uricase and xanthine oxidase, coupled with an enzymethat employs hydrogen peroxide to oxidize a dye precursor such as HRP,lactoperoxidase, or microperoxidase, biotin/avidin, spin labels,bacteriophage labels, stable free radicals, and the like.

V. Pharmaceutical Formulations

Pharmaceutical formulations of a bispecific antibody as described hereinare prepared by mixing such antibody having the desired degree of puritywith one or more optional pharmaceutically acceptable carriers(Remington's Pharmaceutical Sciences, 16th edition, Osol, A. (ed.),(1980)), in the form of lyophilized formulations or aqueous solutions.Pharmaceutically acceptable carriers are generally nontoxic torecipients at the dosages and concentrations employed, and include, butare not limited to: buffers such as phosphate, citrate, and otherorganic acids; antioxidants including ascorbic acid and methionine;preservatives (such as octadecyl dimethylbenzyl ammonium chloride;hexamethonium chloride; benzalkonium chloride; benzethonium chloride;phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol);low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as poly(vinylpyrrolidone); amino acids such asglycine, glutamine, asparagine, histidine, arginine, or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugarssuch as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g. Zn-proteincomplexes); and/or non-ionic surfactants such as polyethylene glycol(PEG). Exemplary pharmaceutically acceptable carriers herein furtherinclude interstitial drug dispersion agents such as solubleneutral-active hyaluronidase glycoproteins (sHASEGP), for example, humansoluble PH-20 hyaluronidase glycoproteins, such as rhuPH20 (HYLENEX®,Baxter International, Inc.). Certain exemplary sHASEGPs and methods ofuse, including rhuPH20, are described in US 2005/0260186 and US2006/0104968. In one aspect, a sHASEGP is combined with one or moreadditional glycosaminoglycanases such as chondroitinases.

Exemplary lyophilized antibody formulations are described in U.S. Pat.No. 6,267,958. Aqueous antibody formulations include those described inU.S. Pat. No. 6,171,586 and WO 2006/044908, the latter formulationsincluding a histidine-acetate buffer.

The formulation herein may also contain more than one active ingredientsas necessary for the particular indication being treated, preferablythose with complementary activities that do not adversely affect eachother. Such active ingredients are suitably present in combination inamounts that are effective for the purpose intended.

Active ingredients may be entrapped in microcapsules prepared, forexample, by coacervation techniques or by interfacial polymerization,for example, hydroxymethylcellulose or gelatin-microcapsules andpoly-(methyl methacrylate) microcapsules, respectively, in colloidaldrug delivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules) or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences,16th edition, Osol, A. (ed.) (1980).

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semi-permeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g. films, or microcapsules.

The formulations to be used for in vivo administration are generallysterile. Sterility may be readily accomplished, e.g., by filtrationthrough sterile filtration membranes.

VI. Therapeutic Methods and Compositions

Any of the bispecific antibodies provided herein may be used intherapeutic methods.

In one aspect, a bispecific antibody for use as a medicament isprovided. In further aspects, a bispecific antibody for use in treatingcancer is provided. In certain embodiments, a bispecific antibody foruse in a method of treatment is provided. In certain embodiments, theinvention provides a bispecific antibody for use in a method of treatingan individual having cancer comprising administering to the individualan effective amount of the bispecific antibody. In one such embodiment,the method further comprises administering to the individual aneffective amount of at least one additional therapeutic agent, e.g., asdescribed below. In further embodiments, the invention provides abispecific antibody for use in removing/killing/lysing cancer cells. Incertain embodiments, the invention provides a bispecific antibody foruse in a method of removing/killing/lysing cancer cells in an individualcomprising administering to the individual an effective of thebispecific antibody to remove/kill/lyse cancer cells. An “individual”according to any of the above embodiments can be a human.

In a further aspect, the invention provides for the use of a bispecificantibody in the manufacture or preparation of a medicament. In oneembodiment, the medicament is for treatment of cancer. In a furtherembodiment, the medicament is for use in a method of treating cancercomprising administering to an individual having cancer an effectiveamount of the medicament. In one such embodiment, the method furthercomprises administering to the individual an effective amount of atleast one additional therapeutic agent, e.g., as described below. In afurther embodiment, the medicament is for removing/killing/lysing cancercells. In a further embodiment, the medicament is for use in a method ofremoving/killing/lysing cancer cells in an individual comprisingadministering to the individual an amount effective of the medicament toremove/kill/lyse cancer cells. An “individual” according to any of theabove embodiments may be a human.

In a further aspect, the invention provides a method for treatingcancer. In one embodiment, the method comprises administering to anindividual having cancer an effective amount of a bispecific antibody.In one such embodiment, the method further comprises administering tothe individual an effective amount of at least one additionaltherapeutic agent, as described below. An “individual” according to anyof the above embodiments may be a human.

In a further aspect, the invention provides a method forremoving/killing/lysing cancer cells in an individual. In oneembodiment, the method comprises administering to the individual aneffective amount of the bispecific antibody to remove/kill/lyse cancercells. In one embodiment, an “individual” is a human.

In a further aspect, the invention provides pharmaceutical formulationscomprising any of the bispecific antibodies provided herein, e.g., foruse in any of the above therapeutic methods. In one embodiment, apharmaceutical formulation comprises any of the bispecific antibodiesprovided herein and a pharmaceutically acceptable carrier. In anotherembodiment, a pharmaceutical formulation comprises any of the bispecificantibodies provided herein and at least one additional therapeuticagent, e.g., as described below.

Antibodies of the invention can be used either alone or in combinationwith other agents in a therapy. For instance, an antibody of theinvention may be co-administered with at least one additionaltherapeutic agent. In certain embodiments, an additional therapeuticagent is a cytotoxic agent or a chemotherapeutic agent.

Such combination therapies noted above encompass combined administration(where two or more therapeutic agents are included in the same orseparate formulations), and separate administration, in which case,administration of the antibody of the invention can occur prior to,simultaneously, and/or following, administration of the additionaltherapeutic agent and/or adjuvant. Antibodies of the invention can alsobe used in combination with radiation therapy.

An antibody of the invention (and any additional therapeutic agent) canbe administered by any suitable means, including parenteral,intrapulmonary, and intranasal, and, if desired for local treatment,intralesional administration. Parenteral infusions includeintramuscular, intravenous, intraarterial, intraperitoneal, orsubcutaneous administration. Dosing can be by any suitable route, e.g.by injections, such as intravenous or subcutaneous injections, dependingin part on whether the administration is brief or chronic. Variousdosing schedules including but not limited to single or multipleadministrations over various time-points, bolus administration, andpulse infusion are contemplated herein.

Antibodies of the invention would be formulated, dosed, and administeredin a fashion consistent with good medical practice. Factors forconsideration in this context include the particular disorder beingtreated, the particular mammal being treated, the clinical condition ofthe individual patient, the cause of the disorder, the site of deliveryof the agent, the method of administration, the scheduling ofadministration, and other factors known to medical practitioners. Theantibody need not be, but is optionally formulated with one or moreagents currently used to prevent or treat the disorder in question. Theeffective amount of such other agents depends on the amount of antibodypresent in the formulation, the type of disorder or treatment, and otherfactors discussed above. These are generally used in the same dosagesand with administration routes as described herein, or about from 1 to99% of the dosages described herein, or in any dosage and by any routethat is empirically/clinically determined to be appropriate.

For the prevention or treatment of disease, the appropriate dosage of anantibody of the invention (when used alone or in combination with one ormore other additional therapeutic agents) will depend on the type ofdisease to be treated, the type of antibody, the severity and course ofthe disease, whether the antibody is administered for preventive ortherapeutic purposes, previous therapy, the patient's clinical historyand response to the antibody, and the discretion of the attendingphysician. The antibody is suitably administered to the patient at onetime or over a series of treatments. Depending on the type and severityof the disease, about 1 μg/kg to 15 mg/kg (e.g. 0.5 mg/kg-10 mg/kg) ofantibody can be an initial candidate dosage for administration to thepatient, whether, for example, by one or more separate administrations,or by continuous infusion. One typical daily dosage might range fromabout 1 μg/kg to 100 mg/kg or more, depending on the factors mentionedabove. For repeated administrations over several days or longer,depending on the condition, the treatment would generally be sustaineduntil a desired suppression of disease symptoms occurs. One exemplarydosage of the antibody would be in the range from about 0.05 mg/kg toabout 10 mg/kg. Thus, one or more doses of about 0.5 mg/kg, 2.0 mg/kg,4.0 mg/kg or 10 mg/kg (or any combination thereof) may be administeredto the patient. Such doses may be administered intermittently, e.g.every week or every three weeks (e.g. such that the patient receivesfrom about two to about twenty, or e.g. about six doses of theantibody). An initial higher loading dose, followed by one or more lowerdoses may be administered. An exemplary dosing regimen comprisesadministering [[add exemplary dosing regimen, if known, e.g., “aninitial loading dose of about 4 mg/kg, followed by a weekly maintenancedose of about 2 mg/kg of the antibody”]]. However, other dosage regimensmay be useful. The progress of this therapy is easily monitored byconventional techniques and assays.

It is understood that any of the above formulations or therapeuticmethods may be carried out using an immunoconjugate of the invention inplace of or in addition to a bispecific antibody.

VII. Articles of Manufacture

In another aspect of the invention, an article of manufacture containingmaterials useful for the treatment, prevention and/or diagnosis of thedisorders described above is provided. The article of manufacturecomprises a container and a label or package insert on or associatedwith the container. Suitable containers include, for example, bottles,vials, syringes, IV solution bags, etc. The containers may be formedfrom a variety of materials such as glass or plastic. The containerholds a composition which is by itself or combined with anothercomposition effective for treating, preventing and/or diagnosing thecondition and may have a sterile access port (for example the containermay be an intravenous solution bag or a vial having a stopper pierceableby a hypodermic injection needle). At least one active agent in thecomposition is an antibody of the invention. The label or package insertindicates that the composition is used for treating the condition ofchoice. Moreover, the article of manufacture may comprise (a) a firstcontainer with a composition contained therein, wherein the compositioncomprises an antibody of the invention; and (b) a second container witha composition contained therein, wherein the composition comprises afurther cytotoxic or otherwise therapeutic agent. The article ofmanufacture in this embodiment of the invention may further comprise apackage insert indicating that the compositions can be used to treat aparticular condition. Alternatively, or additionally, the article ofmanufacture may further comprise a second (or third) containercomprising a pharmaceutically-acceptable buffer, such as bacteriostaticwater for injection (BWFI), phosphate-buffered saline, Ringer's solutionand dextrose solution. It may further include other materials desirablefrom a commercial and user standpoint, including other buffers,diluents, filters, needles, and syringes.

It is understood that any of the above articles of manufacture mayinclude an immunoconjugate of the invention in place of or in additionto a bispecific antibody.

Description of the Sequence Listing:

SEQ ID NO: 01 to 07 and 66 to 67 Sortase motifs

SEQ ID NO: 08 Fc-region nucleophile

SEQ ID NO: 09 to 10 Sortase motif remainders in the conjugate

SEQ ID NO: 11 to 29 Amino acid sequence tag

SEQ ID NO: 30 Human CH2 domain

SEQ ID NO: 31 Human CH3 domain

SEQ ID NO: 32 to 46 Exemplary wild-type and variant antibody heavy chainFc-region polypeptides

SEQ ID NO: 47 to 65 Sequences used in the examples.

EXAMPLES

The following examples are examples of methods and compositions of theinvention. It is understood that various other embodiments may bepracticed, given the general description provided above.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, the descriptions and examples should not be construed aslimiting the scope of the invention.

Materials and Methods

Recombinant DNA Techniques

Standard methods were used to manipulate DNA as described in Sambrook,J., et al., Molecular Cloning: A Laboratory Manual; Cold Spring HarborLaboratory Press, Cold Spring Harbor, New York (1989). The molecularbiological reagents were used according to the manufacturer'sinstructions.

Gene Synthesis

Desired gene segments were prepared by chemical synthesis at GeneartGmbH (Regensburg, Germany). The synthesized gene fragments were clonedinto an E. coli plasmid for propagation/amplification. The DNA sequenceof the subcloned gene fragments were verified by DNA sequencing.

Protein Determination

The protein concentration of purified polyp eptides was determined bydetermining the optical density (OD) at 280 nm, using the molarextinction coefficient calculated on the basis of the amino acidsequence of the polypeptide.

Example 1

Generation of the Expression Plasmids

Description of the Basic/Standard Mammalian Expression Plasmid

Desired proteins were expressed by transient transfection of humanembryonic kidney cells (HEK 293). For the expression of a desiredgene/protein (e.g. full length antibody heavy chain, full lengthantibody light chain, or an Fc-chain containing an oligoglycine at itsN-terminus) a transcription unit comprising the following functionalelements was used:

-   -   the immediate early enhancer and promoter from the human        cytomegalovirus (P-CMV) including intron A,    -   a human heavy chain immunoglobulin 5′-untranslated region        (5′UTR),    -   a murine immunoglobulin heavy chain signal sequence (SS),    -   a gene/protein to be expressed (e.g. full length antibody heavy        chain), and    -   the bovine growth hormone polyadenylation sequence (BGH pA).

Beside the expression unit/cassette including the desired gene to beexpressed the basic/standard mammalian expression plasmid contains

-   -   an origin of replication from the vector pUC 18 which allows        replication of this plasmid in E. coli, and    -   a beta-lactamase gene which confers ampicillin resistance in E.        coli.

Expression plasmids coding for the following polypeptides/proteins wereconstructed:

-   -   Pertuzumab heavy chain variable domain combined with a human        heavy chain constant region of the subclass IgG1 containing a        T366W mutation:

(SEQ ID NO: 47) EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK.

-   -   Pertuzumab light chain variable domain combined with a human        kappa light chain constant region:

(SEQ ID NO: 48) DIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC.

-   -   Trastuzumab heavy chain variable domain combined with a human        heavy chain constant region of the subclass IgG1 containing a        T366S, L368A, and Y407V mutation:

(SEQ ID NO: 49) EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK.

-   -   Trastuzumab light chain variable domain combined with a human        kappa light chain constant region:

(SEQ ID NO: 50) DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC.

-   -   antibody Fab fragment comprising a Pertuzumab heavy chain        variable domain and a human heavy chain constant region 1 (CH1)        of the subclass IgG1 containing a C-terminal GGGSLPETGGSGSHHHHHH        amino acid sequence:

(SEQ ID NO: 51) EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCGGGSLPETGGSGSHHHHHH.

-   -   antibody Fab fragment comprising a Pertuzumab heavy chain        variable domain and a human heavy chain constant region 1 (CH1)        of the subclass IgG1 containing a C-terminal GSLPETGGSGSHHHHHH        sequence:

(SEQ ID NO: 52) EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCGSLPETGGSGSHHHHHH.

-   -   antibody Fab fragment comprising a Pertuzumab heavy chain        variable domain and a human heavy chain constant region 1 (CH1)        of the subclass IgG1 containing a C-terminal LPETGGSGSHHHHHH        sequence:

(SEQ ID NO: 53) EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCLPETGGSGSHHHHHH.

-   -   antibody Fab fragment comprising a Trastuzumab heavy chain        variable domain and a human heavy chain constant region 1 (CH1)        of the subclass IgG1 containing a C-terminal GGGSLPETGGSGSHHHHHH        sequence:

(SEQ ID NO: 54) EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCGGGSLPETGGSGSHHHH HH.

-   -   antibody Fab fragment comprising a Trastuzumab heavy chain        variable domain and a human heavy chain constant region 1 (CH1)        of the subclass IgG1 containing a C-terminal GSLPETGGSGSHHHHHH        sequence:

(SEQ ID NO: 55) EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCGSLPETGGSGSHHHHHH.

-   -   antibody Fab fragment comprising a Trastuzumab heavy chain        variable domain and a human heavy chain constant region 1 (CH1)        of the subclass IgG1 containing a C-terminal LPETGGSGSHHHHHH        sequence:

(SEQ ID NO: 56) EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCLPETGGSGSHHHHHH.

-   -   heavy chain Fc-region polypeptide (human IgGl(CH2-CH3)) with        T366S, L368A, and Y407V mutation containing an N-terminal        GGGDKTHTCPPC sequence:

(SEQ ID NO: 57) GGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

-   -   heavy chain Fc-region polypeptide (human IgG1(CH2-CH3)) with        T366S, L368A, and Y407V mutation containing an N-terminal        GGHTCPPC sequence:

(SEQ ID NO: 58) GGHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

-   -   heavy chain Fc-region polypeptide (human IgG1(CH2-CH3)) with        T366S, L368A, and Y407V mutation containing an N-terminal GGCPPC        sequence:

(SEQ ID NO: 59) GGCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

-   -   heavy chain Fc-region polypeptide (human IgG1(CH2-CH3)) with        T366W mutation containing an N-terminal GGGDKTHTCPPC sequence:

(SEQ ID NO: 60) GGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

-   -   heavy chain Fc-region polypeptide (human IgG1(CH2-CH3)) with        T366W mutations containing an N-terminal GGHTCPPC sequence:

(SEQ ID NO: 61) GGHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

-   -   heavy chain Fc-region polypeptide (human IgG1(CH2-CH3)) with        T366W mutation containing an N-terminal GGCPPC sequence:

(SEQ ID NO: 62) GGCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

Example 2

Transient Expression, Purification and Analytical Characterization

The antibody chains were generated by transient transfection of HEK293cells (human embryonic kidney cell line 293-derived) cultivated in F17Medium (Invitrogen Corp.). For transfection “293-Fectin” TransfectionReagent (Invitrogen) was used. The antibody chains were expressed fromthree different plasmids, coding for a full length heavy chain (eitherPertuzumab-knob, or Trastuzumab-hole), a corresponding full length lightchain, and a heavy chain Fc-region polypeptide containing one of theN-terminal oligoglycine sequences either as knob, or as hole variant.The three plasmids were used at an equimolar plasmid ratio upontransfection. Transfections were performed as specified in themanufacturer's instructions. Antibody Fc-region-containing cell culturesupernatants were harvested seven days after transfection. Supernatantswere stored frozen until purification.

The antibody Fc-region-containing culture supernatants were filtered andpurified by two chromatographic steps. The antibody Fc-regions werecaptured by affinity chromatography using HiTrap MabSelectSuRe (GEHealthcare) equilibrated with PBS (1 mM KH₂PO₄, 10 mM Na₂HPO₄, 137 mMNaC1, 2.7 mM KCl), pH 7.4. Unbound proteins were removed by washing withequilibration buffer, and the antibody Fc-region was recovered with 0.1M citrate buffer, pH 3.0. Immediately after elution the solution wasneutralized to pH 6.0 with 1 M Tris-base, pH 9.0. Size exclusionchromatography on Superdex 200™ (GE Healthcare) was used as secondpurification step. The size exclusion chromatography was performed in 40mM Tris-HCl buffer, 0.15 M NaCl, pH 7.5. The eluted antibody Fc-regionswere concentrated with an Ultrafree-CL centrifugal filter unit equippedwith a Biomax-SK membrane (Millipore, Billerica, Mass.) and stored at−80° C.

The protein concentrations of the antibody Fc-regions were determined bymeasuring the optical density (OD) at 280 nm, using the molar extinctioncoefficient calculated on the basis of the amino acid sequence. Purityand proper antibody Fc-region formation were analyzed by SDS-PAGE in thepresence and absence of a reducing agent (5 mM 1,4-dithiotreitol) andstaining with Coomassie brilliant blue.

Example 3

Transient Expression, Purification and Analytical Characterization ofAntibody Fab Fragments Containing the C-Terminal LPX1TG Motif

The antibody Fab fragments were generated by transient transfection ofHEK293 cells (human embryonic kidney cell line 293-derived) cultivatedin F17 Medium (Invitrogen Corp.). For transfection “293-Fectin”Transfection Reagent (Invitrogen) was used. The antibody Fab fragmentswere expressed from two different plasmids, coding for a full lengthlight chain (either Pertuzumab, or Trastuzumab) and a correspondingtruncated heavy chain containing one of the C-terminal LPX1TG sequences.The two plasmids were used at an equimolar plasmid ratio upontransfection. Transfections were performed as specified in themanufacturer's instructions. Fab fragment-containing cell culturesupernatants were harvested seven days after transfection. Supernatantswere stored frozen until purification.

The Fab fragment containing culture supernatants were filtered andpurified by two chromatographic steps. The Fab fragments were capturedby affinity chromatography using HisTrap HP Ni-NTA columns (GEHealthcare) equilibrated with PBS and 20 mM Imidazole (1 mM KH₂PO₄, 10mM Na₂HPO₄, 137 mM NaCl, 2.7 mM KCl, 20 mM Imidazole), pH 7.4. Unboundproteins were removed by washing with equilibration buffer. Thehistidine-tagged protein was eluted with a 20 mM to 400 mM linearimidazole gradient in PBS (1 mM KH₂PO₄, 10 mM Na₂HPO₄, 137 mM NaCl, 2.7mM KCl, 400 mM Imidazole) in 10 column volumes. Size exclusionchromatography on Superdex 200™ (GE Healthcare) was used as secondpurification step. The size exclusion chromatography was performed in 40mM Tris-HCl buffer, 0.15 M NaCl, pH 7.5. The Fab fragments wereconcentrated with an Ultrafree-CL centrifugal filter unit equipped witha Biomax-SK membrane (Millipore, Billerica, Mass.) and stored at −80° C.

The protein concentrations of the Fab fragments were determined bymeasuring the optical density (OD) at 280 nm, using the molar extinctioncoefficient calculated on the basis of the amino acid sequence. Purityand proper Fab formation were analyzed by SDS-PAGE in the presence andabsence of a reducing agent (5 mM 1,4-dithiotreitol) and staining withCoomassie brilliant blue.

Example 4

Sortase A Mediated Ligation of Antibody Fc-Region and Binding Entity(Fab Fragment)

For the sortase-mediated transpeptidation reaction, N-terminallytruncated Staphylococcus aureus Sortase A was used (Δ₁₋₅₉). The reactionwas performed in a buffer containing 50 mM Tris-HCl, 150 mM NaCl, pH 7.5(Sortase-buffer). In the reaction, a Fab fragment bearing a sortasemotif (LPETG) at its C-terminus of the VH-CH1-heavy chain including noor 2 different connecting short amino acid sequences between theC-terminal end of the VH-CH1 heavy chain ( . . . KSC) and the N-terminusof the sortase motif (LPETGGSGSHHHHHH, SEQ ID NO: 63, GSLPETGGSGSHHHHHH,SEQ ID NO: 64, and GGGSLPETGGSGSHHHHHH, SEQ ID NO: 65) and a one-armedantibody bearing an oligoglycine motif and three different hingesequences (GGCPPC, SEQ ID NO: 8 with X4=P, GGHTCPPC, SEQ ID NO: 66, andGGGDKTHTCPPC, SEQ ID NO: 67, respectively) at its N-terminus of theheavy chain Fc-region polypeptide were linked, resulting in the antibodyFc-region conjugate. To perform the reaction, all reagents were broughtin solution in sortase buffer. In a first step, the antibody Fc-regionand the antibody Fab fragment were mixed, and the reaction was startedby the following addition of Sortase A and 5 mM CaCl₂. The componentswere mixed by pipetting and incubated at 37° C. for 72 h. Subsequently,the reaction was stopped by freezing of the reaction mixture and storageat −20° C. until analysis.

Molar ratio Fab:One-armed antibody:sortase=20:4:1

Results

Three different sequences at the C-terminus of the Fab and at theN-terminus of the antibody respectively were conjugated by Sortase A toobtain nine different combinations of antibody Fc-region conjugates. Theefficiency of the coupling reaction was evaluated at different timepoints. To this end aliquots of the transpeptidation reactions wereanalyzed by SDS-PAGE. The efficiency of ligation was estimateddensitometrically from the SDS PAGE gel. Results after 72 h of reactionare depicted in Table 2 for the respective sequences.

TABLE 2 Conjugation of Fab fragments with one-armed antibodies One armedantibody Fc-region Fab VH-CH1 (OA-Fc-region) heavy chain GGGDKTHTCPPCGGHTCPPC GGCPPC KSCGGGSLPETGGSGSHHHH approx. approx. approx. HH 54% 62%73% KSCGSLPETGGSGSHHHHHH approx. approx. approx. 56% 56% 73%KSCLPETGGSGSHHHHHH approx. approx. approx. 52% 54% 54%

1. A method for producing a bispecific antibody comprising incubating(i) an antibody Fab fragment or a scFv antibody comprising within its 20C-terminal amino acid residues the amino acid sequence LPX1TG (SEQ IDNO: 01, wherein X1is any amino acid residue), (ii) a one-armed antibodyfragment comprising a full length antibody heavy chain, a full lengthantibody light chain, and an antibody heavy chain Fc-region polypeptide,wherein the full length antibody heavy chain and the full lengthantibody light chain are cognate antibody chains complementary to eachother and the pair of variable domains (VH and VL) thereof forms anantigen binding site, wherein the full length antibody heavy chain andthe antibody heavy chain Fc-region polypeptide are covalently linked toeach other via one or more disulfide bonds forming an antibody hingeregion, and wherein the antibody heavy chain Fc-region polypeptide hasan oligoglycine G_(m) (m=2, or 3, or 4, or 5) amino acid sequence at itsN-terminus, and (iii) a Sortase A enzyme, and thereby producing thebispecific antibody.
 2. A method for producing a bispecific antibodycomprising: (i) determining the cell surface makers present in a cellcontaining sample and selecting thereof at least a first cell surfacemarker and a second cell surface marker, (ii) incubating (a) an antibodyFab fragment or a scFv antibody comprising within the 20 C-terminalamino acid residues the amino acid sequence LPX1TG (SEQ ID NO: 01,wherein X1 can be any amino acid residue), wherein the Fab fragment orscFv antibody specifically binds to a first cell surface marker or itsligand, (b) a one-armed antibody fragment comprising a full lengthantibody heavy chain, a full length antibody light chain, and anantibody heavy chain Fc-region polypeptide, wherein the full lengthantibody heavy chain and the full length antibody light chain arecognate antibody chains complementary to each other and the pair ofvariable domains (VH and VL) thereof forms an antigen binding site thatspecifically binds to a second cell surface marker or its ligand,wherein the full length antibody heavy chain and the antibody heavychain Fc-region polypeptide are covalently linked to each other via oneor more disulfide bonds forming an antibody hinge region, and whereinthe antibody heavy chain Fc-region polypeptide has an oligoglycine G_(m)(m=2, or 3, or 4, or 5) amino acid sequence at its N-terminus, and (c) aSortase A enzyme, and thereby producing the bispecific antibody.
 3. Amethod for determining a combination of antigen binding sitescomprising: (i) determining the binding specificity, and/or selectivity,and/or affinity, and/or effector function, and/or in vivo half-life of amultitude of bispecific antibodies prepared by combining (a) each memberof a first multitude of antibody Fab fragments or scFv antibodyfragments wherein each member comprises within its 20 C-terminal aminoacid residues the amino acid sequence LPX1TG (SEQ ID NO: 01, wherein X1is amino acid residue), wherein the Fab fragment or scFv antibodyspecifically binds to a first epitope or antigen, with (b) each memberof a multitude of one-armed antibody fragments comprising a full lengthantibody heavy chain, a full length antibody light chain, and anantibody heavy chain Fc-region polypeptide, wherein the full lengthantibody heavy chain and the full length antibody light chain arecognate antibody chains complementary to each other and the pair ofvariable domains (VH and VL) thereof forms an antigen binding site thatspecifically binds to a second epitope or antigen, wherein the fulllength antibody heavy chain and the antibody heavy chain Fc-regionpolypeptide are covalently linked to each other via one or moredisulfide bonds forming an antibody hinge region, and wherein theantibody heavy chain Fc-region polypeptide has an oligoglycine G_(m)(m=2, or 3, or 4, or 5) amino acid sequence at its N-terminus, and (c) aSortase A enzyme and (ii) choosing the bispecific antibody with suitablebinding specificity, and/or selectivity, and/or affinity, and/oreffector function, and/or in vivo half-life and thereby determining acombination of antigen binding sites.
 4. A bispecific antibody obtainedby a method according to claim
 1. 5. A bispecific antibody comprisingthe amino acid sequence LPX1TG (SEQ ID NO: 01, wherein X1 is any aminoacid residue) in one of its heavy chains.
 6. The method of claim 1,wherein the Fc-region comprises a mutation of the naturally occurringamino acid residue at position 329 and at least one further mutation ofat least one amino acid residue selected from the group consisting ofamino acid residues at position 228, 233, 234, 235, 236, 237, 297, 318,320, 322 and 331 to a different residue, wherein the residues in theFc-region are numbered according to the EU index of Kabat.
 7. Apharmaceutical formulation comprising a bispecific antibody according toclaim
 5. 8. (canceled)
 9. A one-armed antibody fragment comprising afull length antibody heavy chain, a full length antibody light chain,and an antibody heavy chain Fc-region polypeptide, wherein the fulllength antibody heavy chain and the full length antibody light chain arecognate antibody chains complementary to each other and the pair ofvariable domains (VH and VL) thereof forms an antigen binding site,wherein the full length antibody heavy chain and the antibody heavychain Fc-region polypeptide are covalently linked to each other via oneor more disulfide bonds forming an antibody hinge region, and whereinthe antibody heavy chain Fc-region polypeptide has an oligoglycine G_(m)(m=2, or 3, or 4, or 5) amino acid sequence at its N-terminus.
 10. Anantibody Fab fragment or a scFv antibody comprising within its 20C-terminal amino acid residues the amino acid sequence LPX1TG (SEQ IDNO: 01, wherein X1 is any amino acid residue).
 11. A method of treatingan individual having a disease comprising administering to theindividual an effective amount of the bispecific antibody of claim 5.12. A method of making a one armed-Fc-polypeptide (OA-Fc˜polypeptide)conjugate comprising a one armed antibody variant and a polypeptide, themethod comprising: (i) culturing a first host cell comprising nucleicacid(s) encoding the one-armed antibody variant (OA-Fc-Gm) part of theconjugate comprising a pair of full length antibody heavy and chain andits cognate light chain and a heavy chain antibody Fc-region polypeptideunder conditions suitable for expression of the one-armed antibodyvariant (OA-Fc-Gm); (ii) culturing a second host cell comprising nucleicacid(s) encoding a polypeptide part of the conjugate under conditionssuitable for expression of the polypeptide; and (iii) conjugating theOA-Fc-Gm part of the conjugate and the polypeptide part of the conjugateusing Sortase A mediated transpeptidation.
 13. The method of claim 12,further comprising recovering the OA-Fc-Gm from the host cell first orhost cell culture medium and/or recovering the polypeptide from thesecond host cell or host cell culture medium.
 14. Nucleic acid(s)encoding the bispecific antibody of claim
 5. 15. Nucleic acid(s)encoding a bispecific antibody produced by the method of claim
 1. 16. Apharmaceutical formulation comprising a bispecific antibody produced bythe method of claim
 1. 17. A host cell comprising the nucleic acid ofclaim
 14. 18. A host cell comprising the nucleic acid of claim
 15. 19. Amethod of detecting the presence of cancer cells in a biological samplecomprising contacting the biological sample with the bispecific antibodyof claim 5 under conditions permissive for binding of the bispecificantibody to its antigen or antigens and detecting whether a complex isformed between the bispecific antibody and its antigen or antigens. 20.The bispecific antibody of claim 5, wherein X1 is E.